Chemical induction in quiescence in bacteria

ABSTRACT

Quiescence is induced in cells using indole compounds. Expression continues from extra-chromosomal vectors within the cells during quiescence, while chromosomal expression is suppressed. The cells may be used as factories for the production of large amounts of polypeptides of interest, particularly polypeptides which normally have an adverse effect on cell viability or growth. Expression from an extra-chromosomal vector of interest may be monitored, in view of the reduced background expression from the chromosome. Vector copy number may be amplified. Cell cycles may be synchronized.

CROSS REFERENCE TO RELATED APPLICATIONS

This is the U.S. National Stage of International Application No.PCT/GB2006/004751, filed Dec. 18, 2006, which was published in Englishunder PCT Article 21(2), which in turn claims priority to Application GB0525866.0, filed Dec. 20, 2005. Both applications are incorporatedherein in their entirety.

The present invention relates to cells in culture, in particulargeneration of quiescent cells. Quiescent cells have many applications ingene cloning and expression and in synchronisation of cell division.Methods and means are provided for inducing and capitalising onquiescence of cells. The present invention in various aspects andembodiments involves induction of quiescence using indole compounds.

The genetic blueprint is stored, in an encoded form, in deoxyribonucleicacid (DNA) which is a major component of chromosomes in both prokaryoticand eukaryotic organisms. The unit of information in the DNA is thegene. The majority of genes encode polypeptides whose expressionrequires firstly the transcription of a messenger ribonucleic acid(mRNA) and subsequent translation of the mRNA to produce thepolypeptide. A subset of genes is transcribed but not translated.

The development of recombinant DNA technology over the last 30 years hasmade it possible to identify and isolate genes from any organism andexpress their products in a variety of eukaryotic and prokaryotic celltypes, and in recombinant plants and animals. In vitro protein synthesissystems have also been developed. However, considerations of cost andease of handling favour the use of bacteria and among these the mostcommon host organism is the enteric bacterium Escherichia coli. Itsstrengths include its sophisticated genetics, the ability to growrapidly in inexpensive media, and the availability of many customisedcloning vectors (Baneyx, 1999). Inherent shortcomings of bacterialexpression systems include mis-folding of multiple-domain proteins andthe absence of glycosylation, although the latter is a lesser concernwhen proteins are required for research rather than therapeutic use.

To achieve expression of a foreign gene in a bacterium, the gene isfirst inserted into a small, circular DNA molecule known as a cloningvector and then introduced into the bacterium by transformation orelectroporation. Cloning vectors are often derivatives of plasmids;autonomously-replicating DNA circles which are found extensively innatural populations of bacteria. Typically, vectors carry antibioticresistance genes to facilitate selection of vector-containing cells andexpression signals which direct the host bacterium to synthesiseexogenous genes. The copy number of cloning vectors is oftensignificantly higher than the copy number of the natural plasmids fromwhich they were derived.

A number of factors may reduce the efficiency with which the products ofrecombinant genes are expressed in a bacterial host:

-   -   Conventional approaches express cloned genes in actively-growing        cells. The cellular machinery required for transcription and        translation of the recombinant gene is also required for the        expression of genes essential for the growth of the host cell.        There is thus a conflict between the requirements of the        biotechnologist and the bacterium.    -   The metabolic stress imposed by the expression of a recombinant        gene invariably reduces the growth rate and viability of the        host cell. The higher the copy number of the cloning vector, the        greater the effect. Cells which have lost the cloning vector or        have deleted or rearranged the cloned gene will almost        invariably out-grow the original cell-type, reducing yield and        purity of the product.    -   Expression of the cloned gene is simultaneous with the        expression of large numbers of genes located on the host        chromosome. The recombinant product is therefore likely to        represent a relatively small proportion of total cell protein        synthesis, especially if the copy number of the cloning vector        is not very high.

These problems can potentially be avoided, or at least reduced inseverity, if recombinant genes are expressed in non-growing cells. Thedesirability of uncoupling biomass production from the expression ofcloned genes stimulated interest in the basis of bacterial dormancy(Kaprelyants et al., 1993). Little is known of the transition to thedormant state but it can be induced by restricting the nutrient supplythrough glucose, nitrogen or phosphate limitation. Genes which have beenplaced under the control of starvation-inducible promoters can beexpressed in slowly-growing cells. For example, recombinantβ-galactosidase has been expressed from an E. coli promoter which isinduced in response to carbon starvation. Tunner et al. (1992) and Matin(1992) described how placing of the human growth hormone gene downstreamof the cstA promoter stimulated production of the recombinant protein indense, non-growing cultures. A disadvantage of the starvation anddense-culture approaches is the limitation of nutrients which means thatproduct is unlikely to be produced for extended periods.

In 1999 (Rowe & Summers, 1999) reported the development of a method forthe generation of a non-growing but metabolically-active (“quiescent”)E. coli culture. Quiescent Cells (“Q-Cells”) is a recombinant proteinexpression technology which exploits non-growing E. coli as a cellfactory. The concepts underpinning the quiescent cell system came from astudy of plasmid stability which identified a short, untranslatedtranscript which delays the division of E. coli cells containing plasmidmultimers (Patient & Summers, 1993; Sharpe et al., 1999). Growth arrestis achieved under culture conditions where nutrients are not limiting.It offers a radical solution to the much-debated problem of sustainingprotein synthesis in the absence of rapid cell division (Flickinger &Rouse, 1993). Briefly, over-expression of Rcd (a regulatory RNA encodedby plasmid ColE1) in cells in which a cellular component which normallyantagonises quiescence is disrupted, in particular hns205 or rnc14mutant cells, is used to arrest cell growth at a desired cell density,and at a stage when the chromosome is condensed. This results incomplete growth arrest after approximately 3 hours without any need forresource limitation. Cells entering the quiescent state show abnormalnucleoid condensation which results in global down-regulation ofchromosomal gene expression. However, plasmid genes are unaffected andthe metabolic resources of the cell are channelled towards to theexpression of plasmid-borne genes. The main advantages of the Q-Cellsystem are:

-   1. Biomass production halted without nutrient limitation or the use    of growth-inhibitory substances;-   2. Nucleoid condensation leads to preferential expression of plasmid    genes;-   3. Quiescent cells are capable of both de novo transcription and    translation, and they remain metabolically activity for many hours;-   4. Cloning vector copy number is amplified in the non-growing cells.

See also WO97/34996, which is incorporated herein by reference. Theattention of the reader of the present disclosure is drawn specificallyto WO97/34996, where various explanations of and supporting evidence forapplications of quiescent cells are set out, applicable also for thepresent invention.

Research in the Summers laboratory at Cambridge University including aseries of collaborations with academic and commercial laboratories hasconfirmed the potential of the Q-Cell system. It has been demonstratedthat the quiescent state is normally stable for at least 24 hours andthe establishment of quiescence is independent of growth mediumcomposition and culture density. Successful induction of quiescence hasbeen achieved in shake-flask and fermenter cultures at densities up toOD₆₀₀=50. An antibody fragment produced by Q-Cells in fed-batch cultureis folded correctly and secreted into the supernatant at ten times therate seen in non-quiescent cells under equivalent growth conditions(Mukherjee et al., 2004). However, the work has also identified a numberof potential difficulties in using the Q-Cells expression system.

-   1. The Q-Cell expression strain must be freshly constructed for each    use and care is needed to avoid premature expression of Rcd. This    can cause problems for users who are not skilled in microbiology.-   2. Quiescent cultures sometimes “escape” (i.e. they resume growth    and continue into a conventional stationary phase) with the result    that the advantage of the quiescent state is lost.-   3. A heat shock promoter has been used to achieve reliable control    over Rcd expression. This has the disadvantage that recombinant    protein is expressed at 42° C.

The present invention provides a new way to create quiescent bacteria,especially E. coli, which overcomes these difficulties. Using thismethod, a quiescent bacterial culture is simple to establish,independent of temperature, and stable.

Previously, quiescent E. coli have been created by over-expression ofthe Rcd transcript in a cells in which a cellular component whichnormally antagonises quiescence is disrupted. Such disruption may be byvirtue of a mutation in a gene for the cellular component or otherwiseby provision of a suitable genetic background. Preferred has been theuse of hns205 cells or rnc14 cells. For several years the Summerslaboratory has been seeking the target of the Rcd transcript. As notedin Sharpe et al., 1999, the structure of Rcd suggested a hypothesis thatRcd acts as an anti-sense RNA, but the reported failure to find an RNAtarget led to speculation of an alternative mechanism of action withdirect interaction between Rcd and a protein involved in cell divisionor its regulation. None of the experimental results which have now ledto the identification of the target is in the public domain.

We now report for the first time that we have identified tryptophanaseas a potential target, based on analysis of proteins from an E. colicrude lysate which were retained on an Rcd-bound column.

Tryptophanase converts tryptophan to indole which is known to act as asignalling molecule in E. coli (Wang et al., 2001). Cells deficient inthe Rcd target should be resistant to the inhibition of colony formationon solid medium which results from Rcd over-expression. This was shownby us to be the case for a tryptophanase knockout strain of E. coliwhich was insensitive to extremely high levels of Rcd. Subsequently wedemonstrated that an increased level of intracellular indole resultswhen Rcd is expressed in response to multimerization of a cer⁺ plasmid,or when the Rcd is expressed from an independently-regulated promotersuch as P_(lac). Evidence for a direct interaction between Rcd andtryptophanase was provided by our unpublished observation that, in an invitro assay using purified components, Rcd increases the affinity oftryptophanase for its substrate (tryptophan) approximately four-fold.

We have now established that the addition of indole to broth cultures ofE. coli induces quiescence. Experimentation is described below. We havecoined the term “chemical Q cells” or “cQC” to describe cells madequiescent in accordance with the present invention.

A growth-inhibitory effect of indole is disclosed by Journal ofAntibiotics (Tokyo) 1974 27 (12) 987-988.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows growth inhibition of E. coli BW25113 by indole. (opensquares, no indole; closed diamonds, 1 mM indole; closed squares, 2 mMindole; closed triangles, 3 mM indole; open triangles, 4 mM indole; opencircles, 5 mM indole; closed circles, 6 mM indole)

FIG. 2 a shows that indole causes tryptone-water broth cultures of W3110hns205 to enter a quiescent state. (closed diamonds, W3110 0 mM indole;closed squares, W3110 2 mM indole; closed triangles, W3110 4 mM indole;open circles, W3110 hns205 0 mM indole; open triangles, W3110 hns205 2mM indole; open squares, W3110 hns205 4 mM indole)

FIG. 2 b shows that indole causes L-broth cultures of W3110 hns205 toenter a quiescent state. (diamonds, W3110 hns205 0 mM indole; squares,W3110 hns205 2 mM indole; triangles, W3110 hns205 4 mM indole)

FIG. 3 shows that an indole-induced quiescent state can be induced at arange of culture temperatures. (FIG. 3 a (30° C.): triangles, W3110hns205 0 mM indole; squares, W3110 hns205 2 mM indole. FIG. 3 b (42°C.): circles, W3110 hns205 0 mM indole; diamonds, W3110 hns205 2 mMindole.

FIG. 4 shows that indole-induced quiescent cultures of W3110hns205 canexpress the cytokine hGM-CSF. FIG. 4 a: Growth (OD₆₀₀) ofcytokine-producing cultures (closed diamonds, 0 mM indole; closedsquares, 2 mM indole; closed triangles, 4 mM indole). FIG. 4 b: cytokineexpression (ng ml⁻¹ OD⁻¹) in indole-treated cultures (open diamonds, 0mM indole; open squares, 2 mM indole; open triangles, 4 mM indole).

FIG. 5 shows that indole-induced quiescent cultures of W3110hns205 canexpress beta-galactosidase (LacZ). FIG. 5 a: Growth (OD₆₀₀) ofLacZ-producing cultures (closed diamonds, 0 mM indole; closed squares, 2mM indole). FIG. 5 b: LacZ expression in indole-treated cells (opendiamonds, 0 mM indole; open squares, 2 mM indole).

FIG. 6 shows that indole induces quiescence in an rnc mutant of E. coliW3110. (diamonds, 0 mM indole; squares, 2 mM indole).

FIG. 7 shows a comparison of indole- and Rcd-induced quiescence of W3110hns205. (diamonds, W3110 hns205 pcI857ts pUCdelta lacZ+IPTG; triangles,W3110 hns205 pcI857ts pUCdelta lacZ+IPTG+2 mM indole; squares, W3110hns205 pcI857ts pRcd1+IPTG; circles, W3110 hns205 pcI857tspRcd1+IPTG+indole)

FIG. 8 shows the effect of 3 mM indole, isoquinoline, and indoline onW3110hns-205::Tn10. Growth in L-broth containing tetracycline wasmonitored by measuring OD₆₀₀ of culture samples. A control (ethanol,0.6%) was included to ensure that the ethanol used to prepare stocksolutions of indole and indole-related compounds was not affectinggrowth. Data plotted are the mean of two independent assays. (closeddiamonds, 0 mM indole; closed circles, 0 mM indole+ethanol 0.6%; closedsquares, 3 mM indole; open circles, 3 mM isoquinoline; closed triangles,3 mM indoline)

FIG. 9 shows the effect of 3 mM indole, tryptamine, and IAA onW3110hns-205::Tn10. Growth in L-broth containing tetracycline wasmonitored by measuring OD₆₀₀ of culture samples. A control (ethanol,0.6%) was included to ensure that the ethanol used to prepare stocksolutions of indole and indole-related compounds was not affectinggrowth. Data plotted are the mean of two independent assays. (closeddiamonds, 0 mM indole; closed circles, 0 mM indole+ethanol 0.6%; closedsquares, 3 mM indole; open circles, 3 mM IAA; closed triangles, 3 mMtryptamine).

FIG. 10 shows the effect of 3 mM indole, quinoline, and pyrrole onW3110hns-205::Tn10. Growth in L-broth plus tetracycline was monitored bymeasuring OD₆₀₀ of culture samples. A control (ethanol, 0.6%) wasincluded to ensure that the ethanol used to prepare stock solutions ofindole and indole-related compounds was not affecting growth. Dataplotted are the mean of two independent assays (T: tetracycline).(closed diamonds, 0 mM indole; closed circles, 0 mM indole+ethanol 0.6%;closed squares, 3 mM indole; open circles, 3 mM quinoline; closedtriangles, 3 mM pyrrole).

FIG. 11 shows the effect of 3 mM indole, 3-beta-indoleacrylic acid, and1-acetylindoline on W3110hns-205::Tn10. Growth in L-broth plustetracycline was monitored by measuring OD₆₀₀ of culture samples. Acontrol (ethanol, 2.4%) was included to ensure that the ethanol used toprepare stock solutions of indole and indole-related compounds was notaffecting growth. Data plotted are the mean of two independent assays(T: tetracycline). (closed diamonds, 0 mM indole; closed circles, 0 mMindole+ethanol 2.4%; closed squares, 3 mM indole; open circles, 3 mM1-acetylindoline; closed triangles, 3 mM 3-beta-indoleacrylic acid).

The present invention is founded on the discovery that indole caninhibit the growth of cells in broth culture when the cells aredisrupted in a cellular component which normally antagonises quiescence,for example cells that are hns⁻, i.e. lack or have a defective hns gene.The roles of H-NS protein are reviewed by Ussery et al (1994). Thesequence of H-NS is given in: Pon, et al., Mol. Gen. Genet. 212,199-202. Nomenclature has settled on hns but at some places in theliterature the gene is also referred to as: hnsA, bglY, drdX, msyA, osmZand pilG. Bacteria other than E. coli have H-NS-related proteins forwhich sequences have been published, for example S. Flexneri, S.typhimurium, S. marcescens and P. vulgaris. The present invention is notlimited to E. coli and may be applied to any bacterial species, soreferences to hns⁻ cells should be taken as reference to cells defectiveor deficient in the relevant hns like gene in the bacteria of interest.

Equivalent genes or homologues in other bacteria may be identified usingany of a number of available approaches. H-NS-like proteins may beidentified by DNA and/or amino acid sequence similarity to the E. coligene or protein or homologues which have been identified already inother bacteria species (see above).

Nucleic acid and/or amino acid sequence information for HN—S or ahomologue of either may be used in design of nucleic acid molecules forhybridisation experiments to identify equivalent genes or homologues.Hybridisation may involve probing nucleic acid and identifying positivehybridisation under suitably stringent conditions (in accordance withknown techniques) and/or use of oligonucleotides as primers in a methodof nucleic acid amplification, such as PCR. For probing, preferredconditions are those which are stringent enough for there to be a simplepattern with a small number of hybridisations identified as positivewhich can be investigated further. It is well known in the art toincrease stringency of hybridisation gradually until only a few positiveclones remain.

As an alternative to probing, though still employing nucleic acidhybridisation, oligonucleotides designed to amplify DNA sequences may beused in PCR or other methods involving amplification of nucleic acid,using routine procedures. See for instance “PCR protocols; A Guide toMethods and Applications”, Eds. Innis et al, 1990, Academic Press, NewYork.

Preferred amino acid sequences suitable for use in the design of probesor PCR primers are sequences conserved (completely, substantially orpartly) between at least two known or putative homologues. On the basisof amino acid sequence information oligonucleotide probes or primers maybe designed, taking into account the degeneracy of the genetic code,and, where appropriate, codon usage of the organism from the candidatenucleic acid is derived.

Preferred nucleic acid sequences suitable for use in the design ofprobes or PCR primers are sequences conserved (completely, substantiallyor partly) between at least two known or putative homologues.

Assessment of whether or not a PCR product corresponds to an H-NS-likegene may be conducted in various ways. A PCR band from such a reactionmight contain a complex mix of products. Individual products may becloned and each one individually screened for activity.

A further method of using a sequence to identify other homologues is touse computer searches of expressed sequence tag (EST) and other DNAsequence databases.

Wild-type H-NS (the product of the hns gene) antagonises theestablishment and/or maintenance of quiescence in bacterial cells inbroth. Experimental evidence demonstrates with various hns⁻ alleles,especially truncation alleles, that hns⁻ cells in broth culture enterquiescence on expression of Rcd (See WO97/34996) and on treatment withindole. Illustrative truncated alleles include hns-205 andhns_Tn10(N43). In both cases the C-terminal part of the H-NS protein isabsent due to a transposon insertion in the gene. The 205 allele isdescribed in detail in Dersch, et al., Mol. Gen. Genet. (1994) 245,255-259. The N43 strain was given to us by Prof. I. B. Holland,Université Paris-Sud. The hns-206_Amp allele also used is described indetail in Dersch, et al., Mol. Gen. Genet. (1994) 245, 255-259. In thiscase there is no detectable protein product.

As noted, the sequence of E. coli H-NS is given in Pon et al. (1988)Mol. Gen. Genet. 212 199-202. The present invention may utilisedown-regulation of or a mutation in this gene in E. coli or in an alleleor homologue in other bacterium. An allele or homologue may share acertain level of homology with the sequence of E. coli H-NS. Homologymay be at the nucleotide sequence and/or amino acid sequence level. Insome embodiments, the nucleic acid and/or amino acid sequence shareshomology with the sequence encoded by the nucleotide sequence of E. coliH-NS, preferably at least about 50%, or 60%, or 70%, or 80% homology,most preferably at least 90% or 95% homology. The wild-type gene shareswith the E. coli gene the ability to antagonise the establishment and/ormaintenance of quiescence in a bacterial cell, e.g. an E. coli cell, inbroth culture. A mutant of the wild-type that abolishes, wholly orpartially, this ability may be useful in the present invention.

As is well-understood, homology at the amino acid level is generally interms of amino acid similarity or identity. Similarity allows for“conservative variation”, i.e. substitution of one hydrophobic residuesuch as isoleucine, valine, leucine or methionine for another, or thesubstitution of one polar residue for another, such as arginine forlysine, glutamic for aspartic acid, or glutamine for asparagine.Similarity may be as defined and determined by the TBLASTN program, ofAltschul et al. (1990) J. Mol. Biol. 215: 403-10, which is in standarduse in the art. Homology may be over the full-length of the H-NSsequence, or may be over a contiguous sequence of amino acids e.g. about20, 25, 30, 40, 50 or more amino acids compared with the sequence of Ponet al. At the nucleic acid level, homology may be over the full-lengthor may be over a contiguous sequence of nucleotides, e.g. about, 50, 60,70, 75, 80, 90, 100, 120, 150 or more nucleotides.

Those skilled in the art are well aware of methods which can be employedto generate hns⁻ or other mutant strains of E. coli or equivalents forother bacteria. One well-known technique which is particularly suitableis the use of “Pl transduction” (Miller 1972) to movetransposon-inactivated hns. The technique involves introducing into hostcells, via phage P1 infection, a defective hns gene containing aninserted transposon which includes a selectable marker (e.g. a genewhich confers antibiotic resistance). Recombination within cells resultsin the replacement of the wild-type hns gene with thetransposon-inactivated gene. Selection for the marker enablesidentification of successful recombination events. Only when thetransposon is inserted into the host chromosome is the host positive forthe marker. Selected cells are tested for H-NS⁻ phenotype andsuitability for use in the present invention (Rcd sensitivity).

Without wishing to be bound by theory, it can be hypothesized that H-NSis required for recovery of cells from Rcd- or indole-induced growthinhibition, such that over-expression of Rcd or treatment with an indolecompound in an hns mutant background pushes the cells into a non-growingstate from which they have no escape. Disruption of other cellularcomponents which normally antagonise the establishment and/ormaintenance of quiescence in bacterial cells in broth on treatment withindole or an indole compound may be used in embodiments of the variousaspects of the present invention instead of hns⁻.

Mutations which disrupt the antagonistic effect of a cellular componentmay be screened for by indole treatment or over-expressing Rcd inmutagenised wild-type cells then treating the cells (e.g. three to sixhours after indole or Rcd induction) with an antibiotic such aspenicillin or other molecule which kills growing cells but not cellswhich are not growing. Cycling this treatment several times selects forany host cell mutation which increases the severity of Rcd- orindole-mediated growth inhibition.

Another mutation which diminishes an activity which antagonisesRcd-mediated establishment and/or maintenance of quiescence in abacterial cell in broth culture is an rnc mutation, i.e. RNase IIIdeficiency. The effect may be direct or indirect. RNase III or one ormore other endoribonucleases are responsible for Rcd turnover. Reducingthe endoribonuclease activity responsible for degrading Rcd may be usedto increase levels of Rcd expression and tip the balance towardsestablishment of quiescence in accordance with the present invention.RNase III cleaves double-stranded RNA and appears to specificallyrecognize stem-loop structures (Court, 1993). Much work has been done onthe factors which make transcripts more or less sensitive to the enzyme(see, for example, Hjalt and Wagner, 1995). RNase E has been shown tocleave several antisense RNAs including RNAI (Tomcsányi and Apirion,1985) and CopA (Söderbom et al., 1996). The exoribonucleasespolynucleotide phosphorylase (PNPase) and RNase II, also have key rolesin RNA decay (Donovan and Kushner, 1986). Poly (A) polymerase (PcnB),which catalyzes the template-independent sequential addition of AMP tothe 3′-terminal hydroxyl groups of RNA molecules, has also beenimplicated in degradation of RNAI (the replication inhibitor of plasmidColE1). RNAI undergoes PcnB-dependent polyadenylation in vivo and israpidly degraded, subsequent to RNase E cleavage, in the presence ofPcnB and PNPase (Xu et al., 1993). If the pcnB gene is inactivated, theprocessed species is stabilized.

Using a bacterial strain containing a mutation in a cellular componentwhich in wild-type form antagonises the establishment and/or maintenanceof quiescence in broth culture on expression of Rcd or treatment withindole is one approach in accordance with the present invention. Otherways of antagonising the function or activity of such a cellularcomponent include down-regulation of gene expression, e.g. usingantisense technology, a sequence-specific ribozyme or a modified sigmafactor.

The essence of a preferred embodiment of one aspect of the invention isthat by treatment with an indole compound, hns⁻ host cells, or othercells in which an activity which normally antagonises establishment ofquiescence in bacterial cells in broth culture is abolished, wholly orpartially, can in broth culture be switched to a quiescent state inwhich their growth and division is arrested. In this state the cells mayproduce predominantly or only the products of vector-encoded genes andwith the resources required for high-level expression ofextra-chromosomal genes being readily available. This is useful in bothpreparative and analytical synthesis of the products of cloned genes.

The cell may comprise the vector following transformation of the cell oran ancestor thereof.

According to one aspect of the present invention there is provided ahost cell treated with an indole compound, which cell when in brothculture enters quiescence. A preferred embodiment may employ a hns⁻cell. Other cellular backgrounds, such as mutations, which reduce,diminish or decrease, wholly or partially, activity of a cellularcomponent which antagonises the establishment of quiescence in abacterial cell in broth culture on treatment with indole or an indolecompound may be used instead of hns mutant cells. This should be bornein mind when considering the discussion herein that uses hns mutants asa preferred example. Other backgrounds, including other mutations andsystems in which activity of a cellular component is antagonised, e.g.using antisense, ribozyme or other techniques at the disposal of theperson skilled in the art, may be substituted for hns in the discussionherein.

The term “indole” herein, unless context requires, may be used to referto indole itself and may also be used to refer other indole compounds.Useful in the various aspects and embodiments of the present inventionas disclosed herein are indole compounds that share with indole theability to induce quiescence in bacterial cells, such as E. coli,especially in cells which contain a cellular component of whichwild-type activity to antagonise quiescence is disrupted, such as cellsthat are hns⁻ or have a rnc mutation.

Reference to an “indole compound” herein, unless context requires,should be taken to refer to any member of a class of nitrogen-containingheterocyclic compounds that comprise a structural framework based on theindan carbocyclic ring framework, where one of the carbon atoms in theframework is replaced by a nitrogen atom. The indan ring structureconsists of a phenyl or benzene ring fused at one side to the side of afive membered cyclopentane ring (see below).

The term “indan carbocyclic ring framework” herein refers to thecarbocyclic ring structure of indan or indene. At the side where thefive membered ring is fused to the benzene ring, there is an unsaturatedbond. However, the other bonds in the five membered carbocyclic ring maybe saturated, such as in indan, or unsaturated, as in indene. Examplesof indole compounds where the five membered ring is saturated (indoline)or is unsaturated and contains a double bond (3-H indole), in additionto the bond where the benzene ring is fused to the five membered ring,are shown below.

The term indole compound is not limited by the identity of thesubstituents attached to the rings. An indole compound may contain oneor more substituents or may be completely unsubstituted.

More specifically, the term indole compound refers to a class ofcompounds where the nitrogen forms part of the five membered ring. Theterm indole compound may take a narrower meaning by referring to thosecompounds where the nitrogen atom does not form part of the unsaturatedsix membered ring. In such compounds, the nitrogen atom may be bonded tothe benzene ring or positioned so that it is not directly bonded to thebenzene, such as 1H-isoindole where the nitrogen atom is in the2-position, as shown using the standard numbering scheme for the indancarbocyclic framework below.

If the five membered ring contains an unsaturated bond, in addition tothat provided by the fused benzene ring, then the unsaturated bond isposition between the 1,2 positions, such as in 3H-indole, or between the2,3 positions, as in the compound referred to in the art as indole.

The unsaturated bond may form part of a second fused ring, such as inthe indole compound known in the art as carbazole (see below).

The term indole compound is preferred to refer to compounds based on anindan carbocyclic ring framework that does not contain an additionalring fused to this carbocyclic ring framework, such as the secondbenzene ring in carbazole above. In particular embodiments, the termindole compound refers to the subset of compounds where the nitrogenatom itself is outside of and directly bonded to the benzene ring i.e.the nitrogen atom is in the 1 or 3 positions.

The class of compounds represented by the term indole compound as usedherein may be represented by the general formula (I):

whereinX—Y represents

and

-   -   (i) R^(1a), R^(1b), R^(2a), R^(2b), R^(3a), R^(3b), R⁴, R⁵, R⁶        and R⁷ are each independently selected from biologically        compatible substituents; or    -   (ii) R^(2b) and R^(3b) together form double bond or are linked        to form an optionally substituted benzene ring, and R^(1a),        R^(1b), R^(2a), R^(3a), R⁴, R⁵, R⁶ and R⁷ are each independently        selected from biologically compatible substituents; or    -   (iii) in (A), R^(1b) and R^(2a) together form a double bond or        are linked to form an optionally substituted benzene ring, and        R^(1a), R^(2b), R^(3a), R^(3b), R⁴, R⁵, R⁶ and R⁷ are each        independently selected from biologically compatible        substituents.

The term “biologically compatible substituent” as used herein pertainsto an atom or chemical functional group that is covalently bonded to thestructure represent by general formula (I) above at the positionsR^(1a), R^(1b), R^(2a), R^(2b), R^(3a), R^(3b), R⁴, R⁵, R⁶ and R⁷ suchthat the resulting molecule is compatible with the cells retainingability to express genes from extra-chromosomal vectors, even if thereis some toxicity to the cells (especially if at high concentrations).Biologically compatible substituents may be any substituent selectedfrom the list of substituents presented below.

Typically, the term indole compound refers to a class of compoundshaving a double bond in the five membered ring. In particularembodiments this term refers to those compounds where R^(1b) and R^(2a),or R^(2b) and R^(3b) together form a double bond.

More specifically, the term indole compound refers to a class ofcompounds represented by general formula (I) where X—Y is given by thestructural fragment (B).

In particular, biologically compatible substituents include H, halo,hydroxyl, H, halo, hydroxyl, amino, formyl, carboxy, nitro, nitroso,azido, cyano, isocyano, cyanato, isocyanato, thiocyano, isothiocyano,sulfhydryl, sulfonic acid and the optionally substituted substituentsalkyl, aryl, heterocyclyl, ether, acyl, acyl halide, ester, acyloxy,amido, acylamido, thioamido, thioether, sulfonate, sulfone, sulfonyloxy,sulfinyloxy, sulfamino, sulfonamino, sulfinamino, sulfamyl andsulfonamido.

The optionally substituted alkyl group may be an C₁₋₇alkyl, theheterocyclyl group may be C₃₋₂₀heterocyclyl and the aryl group may beC₅₋₂₀aryl. Further biologically compatible substituents are H, halo,hydroxyl, amino, formyl, carboxy, nitro, sulfhydryl, and the optionallysubstituted substituents C₁₋₇alkyl, C₃₋₂₀heterocyclyl, C₅₋₂₀aryl,C₁₋₇alkoxy, C₆₋₁₀aryloxy, acyl, ester (R=C₁₋₇ alkyl or C₆₋₁₀aryl),acyloxy, amido, acylamido, C₁₋₇alkylthio and C₆₋₁₀arylthio.

For substituents that may be optionally substituted, the optionalsubstituents may be selected from halo, hydroxyl, amino, formyl,carboxy, nitro, nitroso, azido, cyano, isocyano, cyanato, isocyanato,thiocyano, isothiocyano, sulfhydryl, sulfonic acid, C₁₋₇alkyl,C₃₋₂₀heterocyclyl, C₅₋₂₀aryl, C₁₋₇alkoxy, C₆₋₁₀aryloxy, acyl, ester(R=C₁₋₇ alkyl or C₆₋₁₀aryl), acyloxy, amido, acylamido, C₁₋₇alkylthio,C₆₋₁₀arylthio, acyl, acyl halide, acyloxy, amido, acylamido, thioamido,sulfonate, sulfone, sulfonyloxy, sulfinyloxy, sulfamino, sulfonamino,sulfinamino, sulfamyl and sulfonamido. More preferred optionalsubstituents are halo, hydroxyl, amino, carboxy, C₁₋₇alkyl, C₅₋₂₀aryl,C₁₋₇alkoxy and C₆₋₁₀aryloxy.

The most preferred substituent for nitrogen (R^(2a) or R^(1a) instructural fragments (A) and (B) respectively, above). H is the mostpreferred substituent for any of R^(1a), R^(1b), R^(2a), R^(2b), R^(3a),R^(3b), R⁴, R⁵, R⁶ and R⁷.

As will be appreciated by the skilled artisan, the above structure isone of many possible resonance structures which may be drawn to depictthe same compound. As used herein, and unless otherwise specified, areference to one structure is to be considered a reference to allpossible corresponding resonance structures.

Phenyl Groups

Examples of phenyl substituents, R⁴ through R⁷, include, but are notlimited to those discussed below under the heading “substituents.”

If the phenyl group has less than the full complement of substituents,they may be arranged in any combination. For example, if the phenylgroup has a single substituent other than hydrogen, it may be in the R⁴,R⁵, R⁶ or R⁷ position. Similarly, if the phenyl group has twosubstituents other than hydrogen, they may be in the R⁴, R⁵, the R⁴, R⁶,the R⁴, R⁷, the R⁵, R⁶, the R⁵, R⁷, or the R⁶, R⁷ positions. If thephenyl group has three substituents other than hydrogen, they may be in,for example, the R⁴, R⁵, R⁶ or the R⁴, R⁵, R⁷, or the R⁴, R⁶, R⁷ or R⁸,R⁶, R⁷ positions.

In one group of embodiments, the phenyl group has only one substituentother than hydrogen, which is in the R⁴, R⁵, R⁶, or R⁷ position.

Adjacent Substituents May be Linked to Form a Ring

The possibility that substituents adjacent to one another, for exampleR⁴ and R⁵ or R^(2b) and R^(3b), together (including the carbon atoms towhich they are attached) form a cyclic structure is not excluded. Forexample, the substituents, R^(2b) and R^(3b), together with the carbonatoms to which they are attached, may form a fused ring structure, suchas the benzene ring found in carbazole (see above). It is desirable thatadjacent substituents do not link to form a cyclic structure.

Chemical Terms

The term “saturated”, as used herein, pertains to compounds and/orgroups which do not have any carbon-carbon or carbon nitrogen double ortriple bonds.

The term “unsaturated”, as used herein, pertains to compounds and/orgroups which have at least one carbon-carbon or carbon-nitrogen doublebond or carbon-carbon triple bond. Compounds and/or groups may bepartially unsaturated or fully unsaturated.

The term “aliphatic”, as used herein, pertains to compounds and/orgroups which are linear or branched, but not cyclic (also known as“acyclic” or “open-chain” groups).

The term “ring”, as used herein, pertains to a closed ring of covalentlylinked ring atoms, which may be an alicyclic ring or an aromatic ring.The term “alicyclic ring,” as used herein, pertains to a ring which isnot an aromatic ring.

The term “carbocyclic ring,” as used herein, pertains to a ring whereinall of the ring atoms are carbon atoms.

The term “carboaromatic ring”, as used herein, pertains to an aromaticring wherein all of the ring atoms are carbon atoms.

The term “heterocyclic ring”, as used herein, pertains to a ring whereinat least one of the ring atoms is a multivalent ring heteroatom, forexample, nitrogen, phosphorus, silicon, oxygen, or sulfur, though morecommonly nitrogen, oxygen, or sulfur. The heterocyclic ring may havefrom 1 to 4 ring heteroatoms.

The term “cyclic compound”, as used herein, pertains to a compound whichhas at least one ring. The term “cyclyl,” as used herein, pertains to amonovalent moiety obtained by removing a hydrogen atom from a ring atomof a cyclic compound.

Substituents

The phrase “optionally substituted,” as used herein, pertains to aparent group which may be unsubstituted or which may be substituted.

Unless otherwise specified, the term “substituted,” as used herein,pertains to a parent group which bears one or more substituents. Theterm “substituent” is used herein in the conventional sense and refersto a chemical moiety which is covalently attached to, or if appropriate,fused to, a parent group. A wide variety of substituents are well known,and methods for their formation and introduction into a variety ofparent groups are also well known.

Examples of substituents are described in more detail below.

Alkyl: The term “alkyl” as used herein, pertains to a monovalent moietyobtained by removing a hydrogen atom from a carbon atom of a hydrocarboncompound having from 1 to 20 carbon atoms (unless otherwise specified),which may be aliphatic or alicyclic, and which may be saturated orunsaturated (e.g., partially unsaturated, fully unsaturated). Thus, theterm “alkyl” includes the sub-classes alkenyl, alkynyl, cycloalkyl,cycloalkyenyl, cylcoalkynyl, etc., discussed below.

In the context of alkyl groups, the prefixes (e.g., C₁₋₄, C₁₋₇, C₁₋₂₀,C₂₋₇, C₃₋₇, etc.) denote the number of carbon atoms, or range of numberof carbon atoms. For example, the term “C₁₋₄alkyl,” as used herein,pertains to an alkyl group having from 1 to 4 carbon atoms. Examples ofgroups of alkyl groups include C₁₋₄alkyl (“lower alkyl”), C₁₋₇alkyl, andC₁₋₂₀alkyl. Note that the first prefix may vary according to otherlimitations; for example, for unsaturated alkyl groups, the first prefixmust be at least 2; for cyclic and branched alkyl groups, the firstprefix must be at least 3; etc.

Examples of (unsubstituted) saturated alkyl groups include, but are notlimited to, methyl (C₁), ethyl (C₂), propyl (C₃), butyl (C₄), pentyl(C₅) and hexyl (C₆).

Examples of (unsubstituted) saturated linear alkyl groups include, butare not limited to, methyl (C₁), ethyl (C₂), n-propyl (C₃), n-butyl(C₄), and n-pentyl (amyl) (C₅).

Examples of (unsubstituted) saturated branched alkyl groups includeiso-propyl (C₃), iso-butyl (C₄), sec-butyl (C₄), tert-butyl (C₄),iso-pentyl (C₅), and neo-pentyl (C₅).

Alkenyl: The term “alkenyl” as used herein, pertains to an alkyl grouphaving one or more carbon-carbon double bonds. Examples of groups ofalkenyl groups include C₂₋₄alkenyl, C₂₋₇alkenyl, C₂₋₂₀alkenyl.

Examples of (unsubstituted) unsaturated alkenyl groups include, but arenot limited to, ethenyl (vinyl, —CH═CH₂), 1-propenyl (—CH═CH—CH₃),2-propenyl (allyl, —CH—CH═CH₂) and isopropenyl (1-methylvinyl,—C(CH₃)═CH₂).

Alkynyl: The term “alkynyl” as used herein, pertains to an alkyl grouphaving one or more carbon-carbon triple bonds. Examples of groups ofalkynyl groups include C₂₋₄alkynyl, C₂₋₇alkynyl and C₂₋₂₀alkynyl.

Examples of (unsubstituted) unsaturated alkynyl groups include, but arenot limited to, ethynyl (ethinyl, —C≡CH) and 2-propynyl (propargyl,—CH₂—C≡CH).

Cycloalkyl: The term “cycloalkyl” as used herein, pertains to an alkylgroup which is also a cyclyl group; that is, a monovalent moietyobtained by removing a hydrogen atom from an alicyclic ring atom of acarbocyclic ring of a carbocyclic compound, which carbocyclic ring maybe saturated or unsaturated (e.g., partially unsaturated, fullyunsaturated), which moiety has from 3 to 20 carbon atoms (unlessotherwise specified), including from 3 to 20 ring atoms. Thus, the term“cycloalkyl” includes the sub-classes cycloalkyenyl and cycloalkynyl. Insome embodiments, each ring has from 3 to 7 ring atoms. Examples ofgroups of cycloalkyl groups include C₃₋₂₀cycloalkyl, C₃₋₁₅cycloalkyl,C₃₋₁₀cycloalkyl, C₃₋₇cycloalkyl.

Examples of cycloalkyl groups include, but are not limited to, thosederived from:

-   -   saturated monocyclic hydrocarbon compounds: cyclopropane (C₃),        cyclobutane (C₄), cyclopentane (C₅), cyclohexane (C₆),        cycloheptane (C₇), methylcyclopropane (C₄), dimethylcyclopropane        (C₅) and methylcyclobutane (C₅);    -   unsaturated monocyclic hydrocarbon compounds: cyclopropene (C₃),        cyclobutene (C₄), cyclopentene (C₅), cyclohexene (C₆),        methylcyclopropene (C₄), dimethylcyclopropene (C₅);    -   saturated polycyclic hydrocarbon compounds: norpinane (C₇),        norbornane (C₇), adamantane (C₁₀), decalin (C₁₀);    -   unsaturated polycyclic hydrocarbon compounds: camphene (C₁₀),        limonene (C₁₀), pinene (C₁₀);    -   polycyclic hydrocarbon compounds having an aromatic ring: indene        (C₉), indane (C₉), tetraline (1,2,3,4-tetrahydronaphthalene)        (C₁₀C), acenaphthene (C₁₂) and fluorene (C₁₃).

Alkylidene: The term “alkylidene,” as used herein, pertains to adivalent monodentate moiety obtained by removing two hydrogen atoms froman aliphatic or alicyclic carbon atom of a hydrocarbon compound havingfrom 1 to 20 carbon atoms (unless otherwise specified). Examples ofgroups of alkylidene groups include C₁₋₂₀alkylidene, C₁₋₇alkylidene,C₁₋₄alkylidene.

Examples of alkylidene groups include, but are not limited to,methylidene (═CH₂), ethylidene (═CH—CH₃), vinylidene (═C═CH₂),isopropylidene (═C(CH₃)₂), cyclopentylidene, and benzylidene (═CH-Ph).

Alkylidyne: The term “alkylidyne” as used herein, pertains to atrivalent monodentate moiety obtained by removing three hydrogen atomsfrom an aliphatic or alicyclic carbon atom of a hydrocarbon compoundhaving from 1 to 20 carbon atoms (unless otherwise specified). Examplesof groups of alkylidyne groups include C₁₋₂₀alkylidyne, C₁₋₇alkylidyne,C₁₋₄alkylidyne.

Examples of alkylidyne groups include, but are not limited to,methylidyne (≡CH), ethylidyne (≡C—CH₃), and benzylidyne (≡C-Ph).

Carbocyclyl: The term “carbocyclyl” as used herein, pertains to amonovalent moiety obtained by removing a hydrogen atom from a ring atomof a carbocyclic compound, which moiety has from 3 to 20 ring atoms(unless otherwise specified). In some embodiments, each ring has from 3to 7 ring atoms.

In this context, the prefixes (e.g., C₃₋₂₀, C₃₋₇, C₅₋₆, etc.) denote thenumber of ring atoms, or range of number of ring atoms. For example, theterm “C₅₋₆carbocyclyl,” as used herein, pertains to a carbocyclyl grouphaving 5 or 6 ring atoms. Examples of groups of carbocyclyl groupsinclude C₃₋₂₀carbocyclyl, C₃₋₁₀carbocyclyl, C₅₋₁₀carbocyclyl,C₃₋₇carbocyclyl, and C₅₋₇carbocyclyl.

Examples of carbocyclic groups include, but are not limited to, thosedescribed above as cycloalkyl groups; and those described below ascarboaryl groups.

Heterocyclyl: The term “heterocyclyl” as used herein, pertains to amonovalent moiety obtained by removing a hydrogen atom from a ring atomof a heterocyclic compound, which moiety has from 3 to 20 ring atoms(unless otherwise specified), of which from 1 to 10 are ringheteroatoms. In some embodiments, each ring has from 3 to 7 ring atoms,of which from 1 to 4 are ring heteroatoms.

In this context, the prefixes (e.g., C₃₋₂₀, C₃₋₇, C₅₋₆, etc.) denote thenumber of ring atoms, or range of number of ring atoms, whether carbonatoms or heteroatoms. For example, the term “C₅₋₆heterocyclyl” as usedherein, pertains to a heterocyclyl group having 5 or 6 ring atoms.

Examples of (non-aromatic) monocyclic heterocyclyl groups include, butare not limited to, those derived from:

-   -   N₁: aziridine (C₃), azetidine (C₄), pyrrolidine        (tetrahydropyrrole) (C₅), pyrroline (e.g., 3-pyrroline,        2,5-dihydropyrrole) (C₅), 2H-pyrrole or 3H-pyrrole (isopyrrole,        isoazole) (C₅), piperidine (C₆), dihydropyridine (C₆),        tetrahydropyridine (C₆), azepine (C₇);    -   O₁: oxirane (C₃), oxetane (C₄), oxolane (tetrahydrofuran) (C₅),        oxole (dihydrofuran) (C₅), oxane (tetrahydropyran) (C₆),        dihydropyran (C₆), pyran (C₆), oxepin (C₇);    -   S₁: thiirane (C₃), thietane (C₄), thiolane (tetrahydrothiophene)        (C₅), thiane (tetrahydrothiopyran) (C₆), thiepane (C₇);    -   O₂: dioxolane (C₅), dioxane (C₆), and dioxepane (C₇);    -   O₃: trioxane (C₆);    -   N₂: imidazolidine (C₅), pyrazolidine (diazolidine) (C₅),        imidazoline (C₅), pyrazoline (dihydropyrazole) (C₅), piperazine        (C₆);    -   N₁O₁: tetrahydrooxazole (C₅), dihydrooxazole (C₅),        tetrahydroisoxazole (C₅), dihydroisoxazole (C₅), morpholine        (C₆), tetrahydrooxazine (C₆), dihydrooxazine (C₆), oxazine (C₆);    -   N₁S₁: thiazoline (C₅), thiazolidine (C₅), thiomorpholine (C₆);    -   N₂O₁: oxadiazine (C₆);    -   O₁S₁: oxathiole (C₅) and oxathiane (thioxane) (C₆); and,    -   N₁O₁S₁: oxathiazine (C₆).

Examples of substituted (non-aromatic) monocyclic heterocyclyl groupsinclude those derived from saccharides, in cyclic form, for example,furanoses (C₅), such as arabinofuranose, lyxofuranose, ribofuranose, andxylofuranse, and pyranoses (C₆), such as allopyranose, altropyranose,glucopyranose and mannopyranose.

Examples of heterocyclyl groups which are also heteroaryl groups aredescribed below with aryl groups.

Aryl: The term “aryl” as used herein, pertains to a monovalent moietyobtained by removing a hydrogen atom from an aromatic ring atom of anaromatic compound, which moiety has from 3 to 20 ring atoms (unlessotherwise specified). In some embodiments, each ring has from 5 to 7ring atoms.

In this context, the prefixes (e.g., C₃₋₂₀, C₅₋₇, C₅₋₆, etc.) denote thenumber of ring atoms, or range of number of ring atoms, whether carbonatoms or heteroatoms. For example, the term “C₅₋₆aryl,” as used herein,pertains to an aryl group having 5 or 6 ring atoms. Examples of groupsof aryl groups include C₃₋₂₀aryl, C₅₋₂₀aryl, C₅₋₁₅aryl, C₅₋₁₂aryl,C₅₋₁₀aryl, C₅₋₇aryl, C₅₋₆aryl, C₅aryl, and C₆aryl.

The ring atoms may be all carbon atoms, as in “carboaryl groups.”Examples of carboaryl groups include C₃₋₂₀carboaryl, C₅₋₂₀carboaryl,C₅₋₁₅carboaryl, C₅₋₁₂carboaryl, C₅₋₁₀carboaryl, C₅₋₇carboaryl,C₅₋₆carboaryl, C₅carboaryl, and C₆carboaryl.

Examples of carboaryl groups include, but are not limited to, thosederived from benzene (i.e., phenyl) (C₆), naphthalene (C₁₀), azulene(C₁₀), anthracene (C₁₄), phenanthrene (C₁₄), naphthacene (C₁₈), andpyrene (C₁₆).

Examples of aryl groups which comprise fused rings, at least one ofwhich is an aromatic ring, include, but are not limited to, groupsderived from indane (e.g. 2,3-dihydro-1H-indene) (C₉), indene (C₉),isoindene (C₉), tetraline (1,2,3,4-tetrahydronaphthalene (C₁₀),acenaphthene (C₁₂), fluorene (C₁₃), phenalene (C₁₃), acephenanthrene(C₁₅), and aceanthrene (C₁₆).

Alternatively, the ring atoms may include one or more heteroatoms, as in“heteroaryl groups.” Examples of heteroaryl groups includeC₃₋₂₀heteroaryl, C₅₋₂₀heteroaryl, C₅₋₁₅heteroaryl, C₅₋₁₂heteroaryl,C₅₋₁₀heteroaryl, C₅₋₇heteroaryl, C₅₋₆heteroaryl, C₅heteroaryl, andC₆heteroaryl.

Examples of monocyclic heteroaryl groups include, but are not limitedto, those derived from:

-   N₁: pyrrole (azole) (C₅), pyridine (azine) (C₆);-   O₁: furan (oxole) (C₅);-   S₁: thiophene (thiole) (C₅);-   N₁O₁: oxazole (C₅), isoxazole (C₅), isoxazine (C₆);-   N₂O₁: oxadiazole (furazan) (C₅);-   N₃O₁: oxatriazole (C₅);-   N₁S₁: thiazole (C₅), isothiazole (C₅);-   N₂: imidazole (1,3-diazole) (C₅), pyrazole (1,2-diazole) (C₅),    pyridazine (1,2-diazine) (C₆), pyrimidine (1,3-diazine) (C₆) (e.g.,    cytosine, thymine, uracil), pyrazine (1,4-diazine) (C₆);-   N₃: triazole (C₅), triazine (C₆); and,-   N₄: tetrazole (C₅).

Examples of heterocyclic groups (some of which are also heteroarylgroups) which comprise fused rings, include, but are not limited to:

-   -   C₉heterocyclic groups (with 2 fused rings) derived from        benzofuran (O₁), isobenzofuran (O₁), indole (N₁), isoindole        (N₁), indolizine (N₁), indoline (N₁), isoindoline (N₁), purine        (N₄) (e.g., adenine, guanine), benzimidazole (N₂), indazole        (N₂), benzoxazole (N₁O₁), benzisoxazole (N₁O₁), benzodioxole        (O₂), benzofurazan (N₂O₁), benzotriazole (N₃), benzothiofuran        (S₁), benzothiazole (N₁S₁), benzothiadiazole (N₂S);    -   C₁₀heterocyclic groups (with 2 fused rings) derived from        chromene (O₁), isochromene (O₁), chroman (O₁), isochroman (O₁),        benzodioxan (O₂), quinoline (N₁), isoquinoline (N₁), quinolizine        (N₁), benzoxazine (N₁O₁), benzodiazine (N₂), pyridopyridine        (N₂), quinoxaline (N₂), quinazoline (N₂), cinnoline (N₂),        phthalazine (N₂), naphthyridine (N₂), pteridine (N₄);    -   C₁₁heterocylic groups (with 2 fused rings) derived from        benzodiazepine (N₂);    -   C₁₃heterocyclic groups (with 3 fused rings) derived from        carbazole (N₁), dibenzofuran (O₁), dibenzothiophene (S₁),        carboline (N₂), perimidine (N₂), pyridoindole (N₂); and,    -   C₁₄heterocyclic groups (with 3 fused rings) derived from        acridine (N₁), xanthene (O₁), thioxanthene (S₁), oxanthrene        (O₂), phenoxathiin (O₁S₁), phenazine (N₂), phenoxazine (N₁O₁),        phenothiazine (N₁S₁), thianthrene (S₂), phenanthridine (N₁),        phenanthroline (N₂), phenazine (N₂).

Heterocyclic groups (including heteroaryl groups) which have a nitrogenring atom in the form of an —NH— group may be N-substituted, that is, as—NR—. For example, pyrrole may be N-methyl substituted, to giveN-methylpyrrole. Examples of N-substitutents include, but are notlimited to C₁₋₇alkyl, C₃₋₂₀heterocyclyl, C₅₋₂₀aryl, and acyl groups.

Heterocyclic groups (including heteroaryl groups) which have a nitrogenring atom in the form of an —N═ group may be substituted in the form ofan N-oxide, that is, as —N(→O)═ (also denoted —N⁺(→O⁻)═). For example,quinoline may be substituted to give quinoline N-oxide; pyridine to givepyridine N-oxide; benzofurazan to give benzofurazan N-oxide (also knownas benzofuroxan).

Cyclic groups may additionally bear one or more oxo (═O) groups on ringcarbon atoms.

Monocyclic examples of such groups include, but are not limited to,those derived from:

-   C₅: cyclopentanone, cyclopentenone, cyclopentadienone;-   C₆: cyclohexanone, cyclohexenone, cyclohexadienone;-   O₁: furanone (C₅), pyrone (C₆);-   N₁: pyrrolidone (pyrrolidinone) (C₅), piperidinone (piperidone)    (C₆), piperidinedione (C₆);-   N₂: imidazolidone (imidazolidinone) (C₅), pyrazolone (pyrazolinone)    (C₅), piperazinone (C₆), piperazinedione (C₆), pyridazinone (C₆),    pyrimidinone (C₆) (e.g., cytosine), pyrimidinedione (C₆) (e.g.,    thymine, uracil), barbituric acid (C₆);-   N₁S₁: thiazolone (C₅), isothiazolone (C₅);-   N₁O₁: oxazolinone (C₅).

Polycyclic examples of such groups include, but are not limited to,those derived from:

-   C₉: indenedione;-   C₁₀: tetralone, decalone;-   C₁₄: anthrone, phenanthrone;-   N₁: oxindole (C₉);-   O₁: benzopyrone (e.g., coumarin, isocoumarin, chromone) (C₁₀);-   N₁O₁: benzoxazolinone (C₉), benzoxazolinone (C₁₀);-   N₂: quinazolinedione (C₁₀); benzodiazepinone (C₁₁);    bezodiazepinedione (C₁₁);-   N₄: purinone (C₉) (e.g., guanine).

Still more examples of cyclic groups which bear one or more oxo (═O)groups on ring carbon atoms include, but are not limited to, thosederived from:

-   -   cyclic anhydrides (—C(═O)—O—C(═O)— in a ring), including but not        limited to maleic anhydride (C₅), succinic anhydride (C₅), and        glutaric anhydride (C₆);    -   cyclic carbonates (—O—C(═O)—O— in a ring), such as ethylene        carbonate (C₅) and 1,2-propylene carbonate (C₅);    -   imides (—C(═O)—NR—C(═O)— in a ring), including but not limited        to, succinimide (C₅), maleimide (C₅), phthalimide, and        glutarimide (C₆);    -   lactones (cyclic esters, —O—C(═O)— in a ring), including, but        not limited to, β-propiolactone, γ-butyrolactone,        δ-valerolactone (2-piperidone), and ε-caprolactone;    -   lactams (cyclic amides, —NR—C(═O)— in a ring), including, but        not limited to, β-propiolactam (C₄), γ-butyrolactam        (2-pyrrolidone) (C₅), δ-valerolactam (C₆), and ε-caprolactam        (C₇);    -   cyclic carbamates (—O—C(═O)—NR— in a ring), such as        2-oxazolidone (C₅);    -   cyclic ureas (—NR—C(═O)—NR— in a ring), such as 2-imidazolidone        (C₅) and pyrimidine-2,4-dione (e.g., thymine, uracil) (C₆).

The above groups, whether alone or part of another substituent, maythemselves optionally be substituted with one or more groups selectedfrom themselves and the additional substituents listed below.

-   Hydrogen: —H. Note that if the substituent at a particular position    is hydrogen, it may be convenient to refer to the compound or group    as being “unsubstituted” at that position.-   Halo: —F, —Cl, —Br, and —I.-   Hydroxy: —OH.-   Ether: —OR, wherein R is an ether substituent, for example, a    C₁₋₇alkyl group (also referred to as a C₁₋₇alkoxy group, discussed    below), a C₃₋₂₀heterocyclyl group (also referred to as a    C₃₋₂₀heterocyclyloxy group), or a C₅₋₂₀aryl group (also referred to    as a C₅₋₂₀aryloxy group), preferably a C₁₋₇alkyl group.-   Alkoxy: —OR, wherein R is an alkyl group, for example, a C₁₋₇alkyl    group. Examples of C₁₋₇alkoxy groups include, but are not limited    to, —OMe (methoxy), —OEt (ethoxy), —O(nPr) (n-propoxy), —O(iPr)    (isopropoxy), —O(nBu) (n-butoxy), —O(sBu) (sec-butoxy), —O(iBu)    (isobutoxy), and —O(tBu) (tert-butoxy).-   Acetal: —CH(OR¹)(OR²), wherein R¹ and R² are independently acetal    substituents, for example, a C₁₋₇alkyl group, a C₃₋₂₀heterocyclyl    group, or a C₅₋₂₀aryl group, preferably a C₁₋₇alkyl group, or, in    the case of a “cyclic” acetal group, R¹ and R², taken together with    the two oxygen atoms to which they are attached, and the carbon    atoms to which they are attached, form a heterocyclic ring having    from 4 to 8 ring atoms. Examples of acetal groups include, but are    not limited to, —CH(OMe)₂, —CH(OEt)₂, and —CH(OMe)(OEt).-   Hemiacetal: —CH(OH)(OR¹), wherein R¹ is a hemiacetal substituent,    for example, a C₁₋₇alkyl group, a C₃₋₂₀heterocyclyl group, or a    C₅₋₂₀aryl group, preferably a C₁₋₇alkyl group. Examples of    hemiacetal groups include, but are not limited to, —CH(OH)(OMe) and    —CH(OH)(OEt).-   Ketal: —CR(OR¹)(OR²), where R¹ and R² are as defined for acetals,    and R is a ketal substituent other than hydrogen, for example, a    C₁₋₇alkyl group, a C₃₋₂₀heterocyclyl group, or a C₅₋₂₀aryl group,    preferably a C₁₋₇alkyl group. Examples ketal groups include, but are    not limited to, —C(Me)(OMe)₂, —C(Me)(OEt)₂, —C(Me)(OMe)(OEt),    —C(Et)(OMe)₂, —C(Et)(OEt)₂, and —C(Et)(OMe)(OEt).-   Hemiketal: —CR(OH)(OR¹), where R¹ is as defined for hemiacetals, and    R is a hemiketal substituent other than hydrogen, for example, a    C₁₋₇alkyl group, a C₃₋₂₀heterocyclyl group, or a C₅₋₂₀aryl group,    preferably a C₁₋₇alkyl group. Examples of hemiacetal groups include,    but are not limited to, —C(Me)(OH)(OMe), —C(Et)(OH)(OMe),    —C(Me)(OH)(OEt), and —C(Et)(OH)(OEt).-   Oxo (keto, -one): ═O.-   Thione (thioketone): ═S.-   Imino (imine): ═NR, wherein R is an imino substituent, for example,    hydrogen, C₁₋₇alkyl group, a C₃₋₂₀heterocyclyl group, or a C₅₋₂₀aryl    group, preferably hydrogen or a C₁₋₇alkyl group. Examples of ester    groups include, but are not limited to, ═NH, ═NMe, ═NEt, and ═NPh.-   Formyl (carbaldehyde, carboxaldehyde): —C(═O)H.-   Acyl (keto): —C(═O)R, wherein R is an acyl substituent, for example,    a C₁₋₇alkyl group (also referred to as C₁₋₇alkylacyl or    C₁₋₇alkanoyl), a C₃₋₂₀heterocyclyl group (also referred to as    C₃₋₂₀heterocyclylacyl), or a C₅₋₂₀aryl group (also referred to as    C₅₋₂₀arylacyl), preferably a C₁₋₇alkyl group. Examples of acyl    groups include, but are not limited to, —C(═O)CH₃ (acetyl),    —C(═O)CH₂CH₃ (propionyl), —C(═O)C(CH₃)₃ (t-butyryl), and —C(═O)Ph    (benzoyl, phenone).-   Carboxy (carboxylic acid): —C(═O)OH.-   Thiocarboxy (thiocarboxylic acid): —C(═S)SH.-   Thiolocarboxy (thiolocarboxylic acid): —C(═O)SH.-   Thionocarboxy (thionocarboxylic acid): —C(═S)OH.-   Imidic acid: —C(═NH)OH.-   Hydroxamic acid: —C(═NOH)OH.-   Ester (carboxylate, carboxylic acid ester, oxycarbonyl): —C(═O)OR,    wherein R is an ester substituent, for example, a C₁₋₇alkyl group, a    C₃₋₂₀heterocyclyl group, or a C₅₋₂₀aryl group, a C₁₋₇alkyl group.    Examples of ester groups include, but are not limited to,    —C(═O)OCH₃, —C(═O)OCH₂CH₃, —C(═O)OC(CH₃)₃, and —C(═O)OPh.-   Acyloxy (reverse ester): —OC(═O)R, wherein R is an acyloxy    substituent, for example, a C₁₋₇alkyl group, a C₃₋₂₀heterocyclyl    group, or a C₅₋₂₀aryl group, preferably a C₁₋₇alkyl group. Examples    of acyloxy groups include, but are not limited to, —OC(═O)CH₃    (acetoxy), —OC(═O)CH₂CH₃, —OC(═O)C(CH₃)₃, —OC(═O) Ph, and    —OC(═O)CH₂Ph.-   Oxycarboyloxy: —OC(═O)OR, wherein R is an ester substituent, for    example, a C₁₋₇alkyl group, a C₃₋₂₀heterocyclyl group, or a    CO₅₋₂₀aryl group, preferably a C₁₋₇alkyl group. Examples of ester    groups include, but are not limited to, —OC(═O)OCH₃, —OC(═O)OCH₂CH₃,    —OC(═O)OC(CH₃)₃, and —OC(═O)OPh.-   Amino: —NR¹R², wherein R¹ and R² are independently amino    substituents, for example, hydrogen, a C₁₋₇alkyl group (also    referred to as C₁₋₇alkylamino or di-C₁₋₇alkylamino), a    C₃₋₂₀heterocyclyl group, or a C₅₋₂₀aryl group, preferably H or a    C₁₋₇alkyl group, or, in the case of a “cyclic” amino group, H and    R², taken together with the nitrogen atom to which they are    attached, form a heterocyclic ring having from 4 to 8 ring atoms.    Amino groups may be primary (—NH₂), secondary (—NHR¹), or tertiary    (—NHR¹R²), and in cationic form, may be quaternary (—⁺NR¹R²R³).    Examples of amino groups include, but are not limited to, —NH₂,    —NHCH₃, —NHC(CH₃)₂, —N(CH₃)₂, —N(CH₂CH₃)₂, and —NHPh. Examples of    cyclic amino groups include, but are not limited to, aziridino,    azetidino, pyrrolidino, piperidino, piperazino, morpholino, and    thiomorpholino.-   Amido (carbamoyl, carbamyl, aminocarbonyl, carboxamide):    —C(═O)NR¹R², wherein R¹ and R² are independently amino substituents,    as defined for amino groups. Examples of amido groups include, but    are not limited to, —C(═O)NH₂, —C(═O)NHCH₃, —C(═O)N(CH₃)₂,    —C(═O)NHCH₂CH₃, and —C(═O)N(CH₂CH₃)₂, as well as amido groups in    which R¹ and R², together with the nitrogen atom to which they are    attached, form a heterocyclic structure as in, for example,    piperidinocarbonyl, morpholinocarbonyl, thiomorpholinocarbonyl, and    piperazinocarbonyl.-   Thioamido (thiocarbamyl): —C(═S)NR¹R², wherein R¹ and R² are    independently amino substituents, as defined for amino groups.    Examples of amido groups include, but are not limited to, —C(═S)NH₂,    —C(═S)NHCH₃, —C(═S)N(CH₃)₂, and —C(═S)NHCH₂CH₃.-   Acylamido (acylamino): —NR¹C(═O)R², wherein R¹ is an amide    substituent, for example, hydrogen, a C₁₋₇alkyl group, a    C₃₋₂₀heterocyclyl group, or a C₅₋₂₀aryl group, preferably hydrogen    or a C₁₋₇alkyl group, and R² is an acyl substituent, for example, a    C₁₋₇alkyl group, a C₃₋₂₀heterocyclyl group, or a C₅₋₂₀aryl group,    preferably hydrogen or a C₁₋₇alkyl group. Examples of acylamide    groups include, but are not limited to, —NHC(═O)CH₃, —NHC(═O)CH₂CH₃,    and —NHC(═O)Ph. R¹ and R² may together form a cyclic structure, as    in, for example, succinimidyl, maleimidyl, and phthalimidyl:

-   Aminocarbonyloxy: —OC(═O)NR¹R², wherein R¹ and R² are independently    amino substituents, as defined for amino groups. Examples of    aminocarbonyloxy groups include, but are not limited to, —OC(═O)NH₂,    —OC(═O)NHMe, —OC(═O)NMe₂, and —OC(═O)NEt₂.-   Ureido: —N(R¹)CONR²R³ wherein R² and R³ are independently amino    substituents, as defined for amino groups, and R¹ is a ureido    substituent, for example, hydrogen, a C₁₋₇alkyl group, a    C₃₋₂₀heterocyclyl group, or a C₅₋₂₀aryl group, preferably hydrogen    or a C₁₋₇alkyl group. Examples of ureido groups include, but are not    limited to, —NHCONH₂, —NHCONHMe, —NHCONHEt, —NHCONMe₂, —NHCONEt₂,    —NMeCONH₂, —NMeCONHMe, —NMeCONHEt, —NMeCONMe₂, and —NMeCONEt₂-   Guanidino: —NH—C(═NH)NH₂.-   Tetrazolyl: a five membered aromatic ring having four nitrogen atoms    and one carbon atom,

-   Imino: ═NR, wherein R is an imino substituent, for example, for    example, hydrogen, a C₁₋₇alkyl group, a C₃₋₂₀heterocyclyl group, or    a C₅₋₂₀aryl group, preferably H or a C₁₋₇alkyl group. Examples of    imino groups include, but are not limited to, ═NH, ═NMe, and ═NEt.-   Amidine (amidino): —C(═NR)NR₂, wherein each R is an amidine    substituent, for example, hydrogen, a C₁₋₇alkyl group, a    C₃₋₂₀heterocyclyl group, or a C₅₋₂₀aryl group, preferably H or a    C₁₋₇alkyl group. Examples of amidine groups include, but are not    limited to, —C(═NH)NH₂, —C(═NH)NMe₂, and —C(═NMe)NMe₂.-   Nitro: —NO₂.-   Nitroso: —NO.-   Azido: —N₃.-   Cyano (nitrile, carbonitrile): —CN.-   Isocyano: —NC.-   Cyanato: —OCN.-   Isocyanato: —NCO.-   Thiocyano (thiocyanato): —SCN.-   Isothiocyano (isothiocyanato): —NCS.-   Sulfhydryl (thiol, mercapto): —SH.-   Thioether (sulfide): —SR, wherein R is a thioether substituent, for    example, a C₁₋₇alkyl group (also referred to as a C₁₋₇alkylthio    group), a C₃₋₂₀heterocyclyl group, or a C₅₋₂₀aryl group, preferably    a C₁₋₇alkyl group. Examples of C₁₋₇alkylthio groups include, but are    not limited to, —SCH₃ and —SCH₂CH₃.-   Disulfide: —SS—R, wherein R is a disulfide substituent, for example,    a C₁₋₇alkyl group, a C₃₋₂₀heterocyclyl group, or a C₅₋₂₀aryl group,    preferably a C₁₋₇alkyl group (also referred to herein as C₁₋₇alkyl    disulfide). Examples of C₁₋₇alkyl disulfide groups include, but are    not limited to, —SSCH₃ and —SSCH₂CH₃.-   Sulfine (sulfinyl, sulfoxide): —S(═O)R, wherein R is a sulfine    substituent, for example, a C₁₋₇alkyl group, a C₃₋₂₀heterocyclyl    group, or a C₅₋₂₀aryl group, preferably a C₁₋₇alkyl group. Examples    of sulfine groups include, but are not limited to, —S(═O)CH₃ and    —S(═O)CH₂CH₃.-   Sulfone (sulfonyl): —S(═O)₂R, wherein R is a sulfone substituent,    for example, a C₁₋₇alkyl group, a C₃₋₂₀heterocyclyl group, or a    C₅₋₂₀aryl group, preferably a C₁₋₇alkyl group, including, for    example, a fluorinated or perfluorinated C₁₋₇alkyl group. Examples    of sulfone groups include, but are not limited to, —S(═O)₂CH₃    (methanesulfonyl, mesyl), —S(═O)₂CF₃ (triflyl), —S(═O)₂CH₂CH₃    (esyl), —S(═O)₂C₄F₉ (nonaflyl), —S(═O)₂CH₂CF₃ (tresyl),    —S(═O)₂CH₂CH₂NH₂ (tauryl), —S(═O)₂Ph (phenylsulfonyl, besyl),    4-methylphenylsulfonyl (tosyl), 4-chlorophenylsulfonyl (closyl),    4-bromophenylsulfonyl (brosyl), 4-nitrophenyl (nosyl),    2-naphthalenesulfonate (napsyl), and    5-dimethylamino-naphthalen-1-ylsulfonate (dansyl).-   Sulfinic acid (sulfino): —S(═O)OH, —SO₂H.-   Sulfonic acid (sulfo): —S(═O)₂OH, —SO₃H.-   Sulfinate (sulfinic acid ester): —S(═O)OR; wherein R is a sulfinate    substituent, for example, a C₁₋₇alkyl group, a C₃₋₂₀heterocyclyl    group, or a C₅₋₂₀aryl group, preferably a C₁₋₇alkyl group. Examples    of sulfinate groups include, but are not limited to, —S(═O)OCH₃    (methoxysulfinyl; methyl sulfinate) and —S(═O)OCH₂CH₃    (ethoxysulfinyl; ethyl sulfinate).-   Sulfonate (sulfonic acid ester): —S(═O)₂OR, wherein R is a sulfonate    substituent, for example, a C₁₋₇alkyl group, a C₃₋₂₀heterocyclyl    group, or a C₅₋₂₀aryl group, preferably a C₁₋₇alkyl group. Examples    of sulfonate groups include, but are not limited to, —S(═O)₂OCH₃    (methoxysulfonyl; methyl sulfonate) and —S(═O)₂OCH₂CH₃    (ethoxysulfonyl; ethyl sulfonate).-   Sulfinyloxy: —OS(═O)R, wherein R is a sulfinyloxy substituent, for    example, a C₁₋₇alkyl group, a C₃₋₂₀heterocyclyl group, or a    C₅₋₂₀aryl group, preferably a C₁₋₇alkyl group. Examples of    sulfinyloxy groups include, but are not limited to, —OS(═O)CH₃ and    —OS(═O)CH₂CH₃.-   Sulfonyloxy: —OS(═O)₂R, wherein R is a sulfonyloxy substituent, for    example, a C₁₋₇alkyl group, a C₃₋₂₀heterocyclyl group, or a    C₅₋₂₀aryl group, preferably a C₁₋₇alkyl group. Examples of    sulfonyloxy groups include, but are not limited to, —OS(═O)₂CH₃    (mesylate) and —OS(═O)₂CH₂CH₃ (esylate).-   Sulfate: —OS(═O)₂OR; wherein R is a sulfate substituent, for    example, a C₁₋₇alkyl group, a C₃₋₂₀heterocyclyl group, or a    C₅₋₂₀aryl group, preferably a C₁₋₇alkyl group. Examples of sulfate    groups include, but are not limited to, —OS(═O)₂OCH₃ and    —SO(═O)₂OCH₂CH₃.-   Sulfamyl (sulfamoyl; sulfinic acid amide; sulfinamide): —S(═O)NR⁸R⁹,    wherein R⁸ and R⁹ are independently amino substituents, as defined    for amino groups. Examples of sulfamyl groups include, but are not    limited to, —S(═O)NH₂, —S(═O)NH(CH₃), —S(═O)N(CH₃)₂,    —S(═O)NH(CH₂CH₃), —S(═O)N(CH₂CH₃)₂, and —S(═O)NHPh.-   Sulfonamido (sulfinamoyl; sulfonic acid amide; sulfonamide):    —S(═O)₂NR⁸R⁹, wherein R⁸ and R⁹ are independently amino    substituents, as defined for amino groups. Examples of sulfonamido    groups include, but are not limited to, —S(═O)₂NH₂, —S(═O)₂NH(CH₃),    —S(═O)₂N(CH₃)₂, —S(═O)₂NH(CH₂CH₃), —S(═O)₂N(CH₂CH₃)₂, and    —S(═O)₂NHPh.-   Sulfamino: —NR⁸S(═O)₂OH, wherein R⁸ is an amino substituent, as    defined for amino groups. Examples of sulfamino groups include, but    are not limited to, —NHS(═O)₂OH and —N(CH₃)S(═O)₂OH.-   Sulfonamino: —NR⁸S(═O)₂R, wherein R⁸ is an amino substituent, as    defined for amino groups, and R is a sulfonamino substituent, for    example, a C₁₋₇alkyl group, a C₃₋₂₀heterocyclyl group, or a    C₅₋₂₀aryl group, preferably a C₁₋₇alkyl group. Examples of    sulfonamino groups include, but are not limited to, —NHS(═O)₂CH₃ and    —N(CH₃)S(═O)₂C₆H₅.-   Sulfinamino: —NR⁸S(═O)R, wherein R⁸ is an amino substituent, as    defined for amino groups, and R is a sulfinamino substituent, for    example, a C₁₋₇alkyl group, a C₃₋₂₀heterocyclyl group, or a    C₅₋₂₀aryl group, preferably a C₁₋₇alkyl group. Examples of    sulfinamino groups include, but are not limited to, —NHS(═O)CH₃ and    —N(CH₃)S(═O)C₆H₅.-   Phosphino (phosphine): —PR₂, wherein R is a phosphino substituent,    for example, —H, a C₁₋₇alkyl group, a C₃₋₂₀heterocyclyl group, or a    C₅₋₂₀aryl group, preferably —H, a C₁₋₇alkyl group, or a C₅₋₂₀aryl    group. Examples of phosphino groups include, but are not limited to,    —PH₂, —P(CH₃)₂, —P(CH₂CH₃)₂, —P(t-Bu)₂, and —P(Ph)₂.-   Phospho: —P(═O)₂.-   Phosphinyl (phosphine oxide): —P(═O)R₂, wherein R is a phosphinyl    substituent, for example, a C₁₋₇alkyl group, a C₃₋₂₀heterocyclyl    group, or a C₅₋₂₀aryl group, preferably a C₁₋₇alkyl group or a    C₅₋₂₀aryl group. Examples of phosphinyl groups include, but are not    limited to, —P(═O) (CH₃)₂, —P(═O) (CH₂CH₃)₂, —P(═O)(t-Bu)₂, and    —P(═O)(Ph)₂.-   Phosphonic acid (phosphono): —P(═O)(OH)₂.-   Phosphonate (phosphono ester): —P(═O)(OR)₂, where R is a phosphonate    substituent, for example, —H, a C₁₋₇alkyl group, a C₃₋₂₀heterocyclyl    group, or a C₅₋₂₀aryl group, preferably —H, a C₁₋₇alkyl group, or a    C₅₋₂₀aryl group. Examples of phosphonate groups include, but are not    limited to, —P(═O) (OCH₃)₂, —P(═O) (OCH₂CH₃)₂, —P(═O) (O-t-Bu)₂, and    —P(═O) (OPh)₂.-   Phosphoric acid (phosphonooxy): —OP(═O)(OH)₂.-   Phosphate (phosphonooxy ester): —OP(═O) (OR)₂, where R is a    phosphate substituent, for example, —H, a C₁₋₇alkyl group, a    C₃₋₂₀heterocyclyl group, or a C₅₋₂₀aryl group, preferably —H, a    C₁₋₇alkyl group, or a C₅₋₂₀aryl group. Examples of phosphate groups    include, but are not limited to, —OP(═O)(OCH₃)₂, —OP(═O) (OCH₂CH₃)₂,    —OP(═O) (O-t-Bu)₂, and —OP(═O) (OPh)₂.-   Phosphorous acid: —OP(OH)₂.-   Phosphite: —OP(OR)₂, where R is a phosphite substituent, for    example, —H, a C₁₋₇alkyl group, a C₃₋₂₀heterocyclyl group, or a    C₅₋₂₀aryl group, preferably —H, a C₁₋₇alkyl group, or a C₅₋₂₀aryl    group. Examples of phosphite groups include, but are not limited to,    —OP(OCH₃)₂, —OP(OCH₂CH₃)₂, —OP(O-t-Bu)₂, and —OP(OPh)₂.-   Phosphoramidite: —OP(OR⁸)—NR⁹ ₂, where R⁸ and R⁹ are phosphoramidite    substituents, for example, —H, a (optionally substituted) C₁₋₇alkyl    group, a C₃₋₂₀heterocyclyl group, or a C₅₋₂₀aryl group, preferably    —H, a C₁₋₇alkyl group, or a C₅₋₂₀aryl group. Examples of    phosphoramidite groups include, but are not limited to,    —OP(OCH₂CH₃)—N(CH₃)₂, —OP(OCH₂CH₃)—N(i-Pr)₂, and —OP(OCH₂CH₂CN)—N    (i-Pr) 2.-   Phosphoramidate: —OP(═O)(OR⁸)—NR⁹ ₂, where R⁸ and R⁹ are    phosphoramidate substituents, for example, —H, a (optionally    substituted) C₁₋₇alkyl group, a C₃₋₂₀heterocyclyl group, or a    C₅₋₂₀aryl group, preferably —H, a C₁₋₇alkyl group, or a C₅₋₂₀aryl    group. Examples of phosphoramidate groups include, but are not    limited to, —OP(═O) (OCH₂CH₃)—N(CH₃)₂, —OP(═O) (OCH₂CH₃)—N(i-Pr)₂,    and —OP(═O) (OCH₂CH₂CN)—N(i-Pr)₂.-   Silyl: —SiR₃, where R is a silyl substituent, for example, —H, a    C₁₋₇alkyl group, a C₃₋₂₀heterocyclyl group, or a C₅₋₂₀aryl group,    preferably —H, a C₁₋₇alkyl group, or a C₅₋₂₀aryl group. Examples of    silyl groups include, but are not limited to, —SiH₃, —SiH₂(CH₃),    —SiH(CH₃)₂, —Si (CH₃)₃, —Si (Et)₃, —Si(iPr)₃, —Si(tBu) (CH₃)₂, and    —Si(tBu)₃.-   Oxysilyl: —Si(OR)₃, where R is an oxysilyl substituent, for example,    —H, a C₁₋₇alkyl group, a C₃₋₂₀heterocyclyl group, or a C₅₋₂₀aryl    group, preferably —H, a C₁₋₇alkyl group, or a C₅₋₂₀aryl group.    Examples of oxysilyl groups include, but are not limited to,    —Si(OH)₃, —Si(OMe)₃, —Si(OEt)₃, and —Si(OtBu)₃.-   Siloxy (silyl ether): —OSiR₃, where SiR₃ is a silyl group, as    discussed above.-   Oxysiloxy: —OSi(OR)₃, wherein OSi(OR)₃ is an oxysilyl group, as    discussed above.

In many cases, substituents are themselves substituted. For example, aC₁₋₇alkyl group may be substituted with, for example:

-   -   hydroxy (also referred to as a hydroxy-C₁₋₇alkyl group);    -   halo (also referred to as a halo-C₁₋₇alkyl group);    -   amino (also referred to as a amino-C₁₋₇alkyl group);    -   carboxy (also referred to as a carboxy-C₁₋₇alkyl group);    -   C₁₋₇alkoxy (also referred to as a C₁₋₇alkoxy-C₁₋₇alkyl group);    -   C₅₋₂₀aryl (also referred to as a C₅₋₂₀aryl-C₁₋₇alkyl group)

Similarly, a C₅₋₂₀aryl group may be substituted with, for example:

-   hydroxy (also referred to as a hydroxy-C₅₋₂₀aryl group);-   halo (also referred to as a halo-C₅₋₂₀aryl group);-   amino (also referred to as an amino-C₅₋₂₀aryl group, e.g., as in    aniline);-   carboxy (also referred to as an carboxy-C₅₋₂₀aryl group, e.g., as in    benzoic acid);-   C₁₋₇alkyl (also referred to as a C₁₋₇alkyl-C₅₋₂₀aryl group, e.g., as    in toluene);-   C₁₋₇alkoxy (also referred to as a C₁₋₇alkoxy-C₅₋₂₀aryl group, e.g.,    as in anisole);-   C₅₋₂₀aryl (also referred to as a C₅₋₂₀aryl-C₅₋₂₀aryl, e.g., as in    biphenyl).    Includes Other Forms

Unless otherwise specified, included in the above are the well knownionic, salt, solvate, and protected forms of these substituents. Forexample, a reference to carboxylic acid (—COOH) also includes theanionic (carboxylate) form (—COO⁻), a salt or solvate thereof, as wellas conventional protected forms. Similarly, a reference to an aminogroup includes the protonated form (—N⁺HR¹R²), a salt or solvate of theamino group, for example, a hydrochloride salt, as well as conventionalprotected forms of an amino group. Similarly, a reference to a hydroxylgroup also includes the anionic form (—O⁻), a salt or solvate thereof,as well as conventional protected forms.

Isomers

Certain compounds may exist in one or more particular geometric,optical, enantiomeric, diasteriomeric, epimeric, atropic,stereoisomeric, tautomeric, conformational, or anomeric forms.

Unless otherwise specified, a reference to a particular compoundincludes all such isomeric forms, including (wholly or partially)racemic and other mixtures thereof. Methods for the preparation (e.g.,asymmetric synthesis) and separation (e.g., fractional crystallisationand chromatographic means) of such isomeric forms are either known inthe art or are readily obtained by adapting the methods taught herein,or known methods, in a known manner.

Note that specifically included in the term “isomer” are compounds withone or more isotopic substitutions. For example, H may be in anyisotopic form, including ¹H, ²H (D), and ³H (T); C may be in anyisotopic form, including ¹²C, ¹³C, and ¹⁴C; O may be in any isotopicform, including ¹⁶O and ¹⁸O; and the like.

Note that, except as discussed below for tautomeric forms, specificallyexcluded from the term “isomers,” as used herein, are structural (orconstitutional) isomers (i.e., isomers which differ in the connectionsbetween atoms rather than merely by the position of atoms in space). Forexample, a reference to a methoxy group, —OCH₃, is not to be construedas a reference to its structural isomer, a hydroxymethyl group, —CH₂OH.Similarly, a reference to ortho-chlorophenyl is not to be construed as areference to its structural isomer, meta-chlorophenyl. However, areference to a class of structures may well include structurallyisomeric forms falling within that class (e.g. C₁₋₇alkyl includesn-propyl and iso-propyl; butyl includes n-, iso-, sec-, and tert-butyl;methoxyphenyl includes ortho-, meta-, and para-methoxyphenyl).

The above exclusion does not pertain to tautomeric forms, for example,keto-, enol-, and enolate-forms, as in, for example, the followingtautomeric pairs: keto/enol (illustrated below), imine/enamine andamide/imino alcohol.

Salts

It may be convenient or desirable to prepare, purify, and/or handle acorresponding salt of the active compound, for example, apharmaceutically-acceptable salt. Examples of pharmaceuticallyacceptable salts are discussed in Berge et al., 1977, “PharmaceuticallyAcceptable Salts,” J. Pharm. Sci., Vol. 66, pp. 1-19.

For example, if the compound is anionic, or has a functional group whichmay be anionic (e.g., —COOH may be —COO⁻), then a salt may be formedwith a suitable cation. Examples of suitable inorganic cations include,but are not limited to, alkali metal ions such as Na⁺ and K⁺, alkalineearth cations such as Ca²⁺ and Mg²⁺, and other cations such as Al⁺³.Examples of suitable organic cations include, but are not limited to,ammonium ion (i.e., NH₄ ⁺) and substituted ammonium ions (e.g., NH₃R⁺,NH₂R₂ ⁺, NHR₃ ⁺, NR₄ ⁺). Examples of some suitable substituted ammoniumions are those derived from: ethylamine, diethylamine,dicyclohexylamine, triethylamine, butylamine, ethylenediamine,ethanolamine, diethanolamine, piperazine, benzylamine,phenylbenzylamine, choline, meglumine, and tromethamine, as well asamino acids, such as lysine and arginine. An example of a commonquaternary ammonium ion is N(CH₃)₄ ⁺.

If the compound is cationic, or has a functional group which may becationic (e.g., —NH₂ may be —NH₃ ⁺), then a salt may be formed with asuitable anion. Examples of suitable inorganic anions include, but arenot limited to, those derived from the following inorganic acids:hydrochloric, hydrobromic, hydroiodic, sulfuric, sulfurous, nitric,nitrous, phosphoric, and phosphorous.

Examples of suitable organic anions include, but are not limited to,those derived from the following organic acids: 2-acetyoxybenzoic,acetic, ascorbic, aspartic, benzoic, camphorsulfonic, cinnamic, citric,edetic, ethanedisulfonic, ethanesulfonic, fumaric, glucheptonic,gluconic, glutamic, glycolic, hydroxymaleic, hydroxynaphthalenecarboxylic, isethionic, lactic, lactobionic, lauric, maleic, malic,methanesulfonic, mucic, oleic, oxalic, palmitic, pamoic, pantothenic,phenylacetic, phenylsulfonic, propionic, pyruvic, salicylic, stearic,succinic, sulfanilic, tartaric, toluenesulfonic, and valeric. Examplesof suitable polymeric organic anions include, but are not limited to,those derived from the following polymeric acids: tannic acid,carboxymethyl cellulose.

Unless otherwise specified, a reference to a particular compound alsoinclude salt forms thereof.

Solvates

It may be convenient or desirable to prepare, purify, and/or handle acorresponding solvate of the active compound. The term “solvate” is usedherein in the conventional sense to refer to a complex of solute (e.g.,active compound, salt of active compound) and solvent. If the solvent iswater, the solvate may be conveniently referred to as a hydrate, forexample, a mono-hydrate, a di-hydrate, a tri-hydrate, etc.

Unless otherwise specified, a reference to a particular compound alsoinclude solvate forms thereof.

Various preferred aspects and embodiments of the present inventionemploy one or more indole compounds selected from the group consistingof isoquinoline, 3-β-indoleacrylic acid, quinoline, indoline andtryptamine.

Compounds that assist the bacterial stress response, such as indoleacetic acid (IAA), may be excluded (Bianco et al., 2006), also pyrrole.

A 0.5 M stock solution of indole may conveniently be prepared in ethanol(0.585 g in 10 ml absolute ethanol, although other stock concentrationsand other solvents are possible). A volume of the indole stock solutionappropriate to achieve the desired final concentration may be addeddirectly to treat cells, for example in a batch culture of cells grownin either shake-flask or a fermenter. For cells grown in fed-batchfermenter culture an appropriate volume of indole stock solution may beintroduced via the feed.

In shake-flask culture we have shown that an appropriate finalconcentration of indole to induce quiescence of E. coli cells is 2 mM.If too little indole is added the cells do not enter quiescence, and iftoo much is added growth may cease immediately (FIGS. 2 a, 2 b, 4 a)with undesirable consequences for the protein synthetic capacity of theculture (FIG. 4 b). It is possible that the concentration of indolerequired to induce quiescent cells with optimum protein syntheticcapacity may vary with the species of bacterium and the growthconditions (growth medium composition, temperature etc). The appropriateconcentration may be established by testing the effect of a range ofindole concentrations and observing their effect on cell growth and theproduction of an appropriate test protein.

The cell may be transformed with a heterologous gene of interest forexpression. “Transformation” refers to any means of introduction ofnucleic acid into a cell. A heterologous gene for expression in the cellmay be cloned into an expression vector or it may be introduceddirectly, as discussed further below.

The heterologous (or “exogenous” or “foreign”) gene of interest may beinducible or constitutively expressed. The heterologous gene product maybe one which has a toxic effect on the cell, particularly one whichadversely affects viability or cell growth and/or division. Theheterologous gene may be a non-E. coli gene, or if a non-E. coli hostcell is employed a gene not of that host, and it may be a eukaryoticgene e.g. mammalian.

In broth culture cells can in accordance with the invention be grown upand treated with an indole compound to induce a quiescent state in whichexpression of genes on the condensed chromosomal DNA is eliminated, orreduced, compared with prior to indole treatment. Expression of plasmidvector borne genes proceeds in these cells.

Expression of a gene product which has an adverse effect on cell growthand/or division, for example one which interferes with replication ofthe bacterial chromosome, transcription of one or more genes essentialto the growth and division of the host cell, or disrupts one or moreother vital processes, is less likely to have an adverse effect onquiescent (non-growing) cells than growing cells.

According to another aspect, the present invention provides a method ofexpressing a gene heterologous to a cell, comprising:

-   (a) growing cells, e.g. hns⁻ cells, in broth culture, the cells    containing an extra-chromosomal heterologous gene;-   (b) treating the cells with an indole compound and causing or    allowing expression of the heterologous gene.

The method may comprise a step of introducing a vector comprisingnucleic acid encoding the gene of interest with suitable controlelements for transcription and translation.

In various embodiments, the invention provides methods as set out in theclaims.

Following production of the heterologous gene product, the method mayinclude any number of conventional purification steps (Harris & Angal,1989).

The expression product may be isolated and/or purified from cells fromthe culture or from the broth culture medium. It is conventional in theart to provide recombinant gene products as a fusion with a “signalsequence” which causes secretion of the product into the growth medium,to facilitate purification. This is one possibility amongst the manyknown to those skilled in the art.

An isolated and/or purified expression product may be modified and theexpression product or a modified form thereof may be formulated into acomposition which includes at least one additional component, such as apharmaceutically acceptable excipient, carrier, buffer, stabiliser orother materials well known to those skilled in the art. Such materialsshould, for a pharmaceutical composition, be non-toxic and should notinterfere with the efficacy of the active ingredient. The precise natureof the carrier or other material will depend on the route ofadministration.

An expression product may be modified for example by chemicalderivatisation or cross-linking to one or more other molecules,including peptides, polypeptides, labelling molecules.

A chemical moiety may be introduced at a specific chemically modifiableresidue or residues. For instance, a cysteine residue may be availablefor chemical modification via its thiol group. Other chemicallymodifiable amino acids include lysine, glutamate, histidine andtyrosine. Covalent modification allows a wide variety of moieties to beincorporated, particularly reporter groups or cofactors for catalysis.This allows the interaction of large organic groups such as thefluorescent reporter group, 7-nitrobenz-2-oxa-1,3-diazole (NBD). Otherlarge groups such as the flavin cofactors for catalysis, FMN and FAD maybe incorporated.

There are other possible ways of modifying a polypeptide. There are anumber of amino acid residues which may be specifically derivatizedusing molecules containing specific functional groups. For instance,amino groups may be modified with N-hydroxysuccinimide esters, carboxylgroups with carbodiimides, histidines and cysteines with halomethylketones, arginine with glyoxals (see e.g. A. R. Fersht, Enzyme Structureand Mechanism 2nd edn, 1985 pp 248-251, W.H. Freeman, New York).

Some reagents which may be used to modify specific amino-acid residuesare given by T. Imoto and H. Yamada in “Protein Function: a PracticalApproach”, pp 247-277, 1989. To introduce specific functional groupsinto polypeptides the reactive group of these reagents may be combinedwith the functional group in a modifying reagent. For instance, if it isdesired to modify a protein with the fluorophore7-amino-4-methylcoumarin-3-acetic acid, the N-hydroxysuccinimidyl esterof the molecule may be used to modify amino groups, whereasN-[6-(-amino-4-methylcoumarin-3-acetamido)hexyl]-3′-(2′-pyridyldithio)propionamidemay be used to modify cysteine groups.

Another possible methodology is to use transglutaminase which catalyzesan acyl-transfer reaction between the gamma-carboxyamide group ofglutamine residues and primary amines (E. Bendixen et al, J. Biol. Chem.26821962-21967, 1993; K. N. Lee et al Biochim. Biophys. Acta 1202 1-61993; T. Kanaji et al J. Biol. Chem. 268 11565-11572 1993). This enzymecould therefore introduce amino acid residues from a peptide into aglutamine residue through a peptide lysine epsilon amino group or into alysine group via a peptide glutamine group. The enzyme could alsocatalyse derivatization of glutamine residues with a primary amine.

A further approach is to introduce chemical moieties to either the N orC terminus of a polypeptide using reverse proteolysis or chemicalconjugation or a combination of the two (I. Fisch et al, Bioconj. Chem.3, 147-153, 1992; H. F. Gaertner et al, Bioconjug. Chem. 3, 262-268,1992; H. F. Gaertner et al, J. Biol. Chem. 269, 7224-7230, 1994; J.Bongers et al, Biochim. Biophys. Acta, 50, S57-162, 1991; R. Offord,Protein Engineering, 4, 709-710, 1991). These methods have been used tointroduce non-encoded elements to protein and peptide molecules.

Examples of fluorophores which may be introduced are fluorescein,phycoerythrin, coumarin, NBD, Texas Red™ and chelated lanthanide ions.Examples of catalytic groups which may be introduced are flavin adeninedinucleotide (FAD), flavin mononucleotide (FMN), cytochromes andchelated metal ions such as zinc and copper.

Systems for cloning and expression of a polypeptide in a variety ofdifferent host cells are well known.

Suitable vectors can be chosen or constructed, containing appropriateregulatory sequences, including promoter sequences, terminators,polyadenylation sequences, enhancer sequences, marker genes and othersequences as appropriate. Vectors may be plasmids, viral e.g. ‘phage, orphagemid, as appropriate. For further details see, for example MolecularCloning: a Laboratory Manual: 3rd edition, Sambrook and Russell, 2001,Cold Spring Harbor Laboratory Press. Many known techniques and protocolsfor manipulation of nucleic acid, for example in preparation of nucleicacid constructs, mutagenesis, sequencing, introduction of DNA into cellsand gene expression, and analysis of proteins, are described in detailin Protocols in Molecular Biology, Second Edition, Ausubel et al. eds.,John Wiley & Sons, 1992.

For bacterial cells, suitable techniques for introducing nucleic acid(“transformation”) may include calcium chloride transformation,electroporation and transfection using bacteriophage.

The introduction may be followed by causing or allowing expression fromthe nucleic acid, e.g. by culturing host cells under conditions forexpression of the gene.

The term “inducible” as applied to a gene or more particularly apromoter is well understood by those skilled in the art. In essence,expression under the control of an inducible promoter is “switched on”or increased in response to an applied stimulus. The nature of thestimulus varies between promoters. Some inducible promoters cause littleor undetectable levels of expression (or no expression) in the absenceof the appropriate stimulus. Other inducible promoters cause detectableconstitutive expression in the absence of the stimulus. Whatever thelevel of expression is in the absence of the stimulus, expression fromany inducible promoter is increased in the presence of the correctstimulus. The desirable situation is where the level of expressionincreases upon application of the relevant stimulus by an amounteffective to alter a phenotypic characteristic. Thus an inducible (or“switchable”) promoter may be used which causes a basic level ofexpression in the absence of the stimulus which level is too low tobring about a desired phenotype (and may in fact be zero). Uponapplication of the stimulus, expression is increased (or switched on) toa level which brings about the desired phenotype.

For use in bacterial systems, many inducible promoters are known (Oldand Primrose, 1994). Common examples include P_(lac) (IPTG), P_(tac)(IPTG), lambdaP_(R) (loss of CI repressor), lambdaP_(L) (loss of CIrepressor), P_(trc) (IPTG), P_(trp) (IAA). The inducing agent is shownin brackets after each promoter.

The use of a quiescent cell system in large scale fermenters may haveone or more advantages, such as a higher yield of product, a greaterpurity of product due to lower levels of contamination by host geneproducts, fewer problems of structural and segregational instabilitybecause of the reduced stress on the host cells.

Generally in the art, problems arise when attempting to monitor theproducts of vector-borne genes, due to the high background ofchromosomal gene products. To circumvent these problems, “minicells” and“maxicells” have been used. Both approaches require the use of hostcells carrying specific mutations. Minicells are chromosome-less cellsproduced as a result of asymmetric deposition of the septum (the “celldivider”) during cell division. Minicells have to be separatedphysically from chromosome-containing cells, typically by sucrosegradient centrifugation; a time consuming and technically-demandingprocedure. Maxicells carry chromosomal mutations which make themUV-sensitive and are irradiated to fragment the bacterial chromosome;plasmids survive by virtue of their high numbers and their small size.The majority of protein synthesis is therefore from plasmid genes.

Use of the present invention offers a simple and effective alternativeto minicells and maxicells. According to another aspect of the inventionthere is a method of monitoring the production of protein by expressionfrom an extra-chromosomal vector of interest, comprising introducing thevector into host cells and growing the cells in culture, treating thecells with an indole compound to induce quiescence, causing or allowingexpression from the vector of interest and determining the expressionfrom the vector. As discussed at length already above, the cells may behns⁻ or be otherwise sensitive to establishment of quiescence ontreatment with indole in broth culture.

Expression at the polypeptide level may be determined by introducing asuitable label into the cells and determining the incorporation of thelabel into produced peptides or polypeptides. The method may compriseintroducing the label into the culture, causing or allowing expressionfrom the vector of interest, lysing cells from the culture, running thelysate on an SDS-polyacrylamide gel and observing the labelled proteinon the gel.

The labelled amino acid may be ³⁵S-methionine. Observation ofradiolabelled proteins may be by autoradiography.

Expression at the mRNA level may be similarly determined by using asuitable label.

The vector of interest may in principle be introduced into the cellsbefore or after treatment with an indole compound. However, it ispreferred that the vector be introduced before indole compoundtreatment.

After quiescence is induced, only extra-chromosomal genes within thecells, such as on vectors such as plasmids, will be expressed, orpredominantly only these genes. The condensation of the chromosomal DNAupon indole treatment reduces or eliminates expression of chromosomalgenes. The protein which can be observed by means of the label will bethat produced after “shut-down” of the chromosomes, i.e. that encoded bynucleic acid retained extra-chromosomally in the cells.

One aspect of the present invention is a method to amplify the copynumber of plasmid cloning vectors when cells enter the quiescent state.This reduces the metabolic load imposed on cells during the growth phaseand increases the copy number of the vector (and thus of the productgene) during the quiescent state. Many plasmid replication controlsystems are described by the “+n” model which states that for a plasmidwith an average copy number of n in a new-born cell, an average of nreplication events will occur per unit time (which is the generationtime in a steady state culture), irrespective of actual copy number(Nielsen and Molin, 1984). We have discovered that for cells in thequiescent state, replication of some plasmids continues, despite thecessation of cell growth and division. It is thus possible to employ alow copy number vector which places the minimum stress on its host cellsduring the growth phase of the culture but which increases in copynumber during and after entry into quiescence.

Many scientific investigations, including in vivo studies of DNAreplication and cell division, are greatly assisted by synchronising thecells within culture so that all cells undergo division at the sametime. Existing procedures are technically-demanding or unreliable,involving the isolation of new-born cells by virtue of their size orusing a bacteriostatic agent (e.g. the antibiotic chloramphenicol) toblock a key process such as the initiation of chromosomal DNAreplication and removing the agent by washing after growth has beenhalted. The reversibility of the block of cell division in accordancewith the present invention provides a simple and effective way toachieve synchronisation of cell division and chromosomal DNAreplication.

Rcd blocks growth at a specific stage in the cell cycle [Patient &Summers, 1993]; chromosome partitioning is complete but septation hasnot occurred. The effect is reversible for cells within the populationwithout serious loss of cell viability, subject to how long the culturehas been in quiescence. Reversing of quiescence induced by means of anindole compound in accordance with the present invention may be employedin aspects and embodiments of the present invention.

According to a further aspect of the present invention there is provideda method of synchronising cell cycles of cells in broth culture,comprising inducing quiescence within (e.g.) hns⁻ cells in broth cultureby treatment of the cells with an indole compound, incubating the cellsfor a time to achieve quiescence, which may for example be equivalent toa further cell cycle, one and half cell cycles or two cell cycles atleast, and removing the indole compound, thereby allowing the cells toexit quiescence.

The present invention further encompasses use of hns⁻ cells or rnc cellsin any of the methods described and vectors specially adapted for use inany of the methods.

The use of indole or other indole compound as disclosed for treatingbacterial cells to induce quiescence represents an aspect of the presentinvention.

As noted, the cells made quiescent in accordance with the variousaspects and embodiments of the present invention, “chemical Q cells”(cQC) may be used in a number of ways, not limited to but including thefollowing.

Cultures of strains, e.g. wherein a cellular component which normallyantagonises quiescence is disrupted, for example hns205 or rnc14 mutantstrains, made quiescent by the addition of indole or other indolecompound to the culture medium (cQC) may be used for recombinant proteinexpression in small-scale shake-flask culture, or in batch or fed-batchfermenter culture. Cells may be grown in any appropriate culture medium.

Recombinant protein may be expressed from any suitable expression vectorat any convenient culture temperature. If the recombinant protein geneis expressed from a promoter up-regulated in response to indole, theproduct may be expressed in cQC without the need for an inducing signal(compare this with the use of the lac operon promoter, P_(lac), whichrequires the addition of IPTG for induction). One such promoter is thatof E. coli gene mdtE, which shows a 22-fold elevation of expression inresponse to 2 mM indole (Hirakawa et al., 2005).

We have further devised an approach to expression in quiescent cellsthat we term “c-QED” (“chemical Quiescent Cells, Expression Direct”).

This application permits the rapid expression of proteins in bacterialcells without the need for inserting the product gene into an expressionvector. It was developed initially for use with the Rcd-inducedquiescent cell system but is useful with chemical Quiescent Cells. Inthis method the product gene, incorporated into a direct expressioncassette, is introduced into the chemical Quiescent Cells by, forexample, transformation or electroporation leading to immediateexpression of the product. The direct expression cassette may be aproduct gene incorporated into a circular or linear DNA molecule. ThisDNA molecule may be a plasmid if it is capable of autonomous replicationbut this is not necessary for the c-QED system. The expression cassettemay be generated by PCR, using appropriate primers to amplify the geneof interest. In this case it is necessary that the ends of the PCRproduct are protected from exonuclease-mediated degradation and thatappropriate transcription and translation control signals are adjacentto the gene of interest. These objectives may both be achieved byligating hairpin-loop DNA molecules onto the ends of the PCR product.The hairpin-loop adjacent to sequences coding for the N-terminus of therecombinant protein should include a promoter and a ribosome bindingsite (RBS). There is no need for such sequences in the hairpin-loopligated to the end of the PCR product adjacent to sequences encoding theC-terminus of the recombinant protein but a transcription terminatorsequence may be included if required. This approach enables the desiredprotein to be expressed directly from a PCR product, without the needfor insertion into an expression vector. It is suitable for small-scale,high-throughput approaches where small amounts of large numbers ofrecombinant proteins are required. It may also be used for the synthesisof proteins which are toxic to their bacterial host. Using conventionalexpression systems the gene encoding the toxic protein must be expressedfrom a promoter which can be tightly repressed up to the time whenprotein expression is required. Using c-QED this difficulty is avoidedbecause the gene encoding the toxic protein is only introduced into thequiescent cells when its expression is required. It is thus possible toexpress a highly toxic gene from an constitutively active promoter.

Various further aspects and embodiments will be apparent to thoseskilled in the art in the light of the present disclosure, including thefollowing experimentation. All documents mentioned anywhere herein areincorporated by reference.

Experimentation

Tryptophanase is Targeted by Rcd

As noted already above, we have shown that a tryptophanase knockoutstrain of E. coli was insensitive to extremely high levels of Rcd, beingresistant to the inhibition of colony formation on solid medium whichresults from Rcd over-expression. This provided indication that Rcdtargets trytophanase. Subsequently we have demonstrated that anincreased level of intracellular indole results when Rcd is expressed inresponse to multimerization of a cer⁺ plasmid, or when the Rcd isexpressed from an independently-regulated promoter such as P_(lac).Evidence for a direct interaction between Rcd and tryptophanase wasprovided by our unpublished observations that, (i) in an in vitro assayusing purified components, Rcd increases the affinity of tryptophanasefor its substrate (tryptophan) approximately four-fold and (ii) usingcolumn chromatography, tryptophanase is retained by a column to whichRcd has been linked covalently.

Generation of Quiescent E. coli By Exogenous Indole

We have now established that the addition of indole to broth cultures ofE. coli inhibits growth (FIG. 1). For E. coli strain BW25113 cultured inL-broth, growth inhibition was not evident at 1 mM indole but partialinhibition was apparent at 2 and 3 mM. Growth inhibition was severe atconcentrations of 4 mM or above.

We found that the response of E. coli W3110 cultured in tryptone waterbroth was qualitatively similar to BW25113 in L-broth (FIG. 2). 2 mMindole caused partial growth inhibition while 4 mM indole inhibitedgrowth severely.

An hns205 mutant of W3110 displayed a different response to indole fromits wild-type parent strain (FIG. 2). In tryptone water broth containing2 mM indole at 37° C., the growth rate of W3110 hns205 declined overapproximately 6 hours before entering a non-growing (quiescent) state.We refer to the indole-induced non-growing state of hns205 mutant cellsas chemically-induced quiescent cells (cQC).

Entry into the quiescent state is temperature-independent. We were ableto induce quiescence by the addition of 2 mM indole at 30° C. and 42° C.(FIG. 3), as well as at 37° C. (FIG. 2). In each case the quiescentstate was stable and persisted for at least 24 hours. The phenomenon of“escape” was not witnessed when working with indole-induced quiescentcultures.

To demonstrate the capacity of indole-induced quiescent E. coli for denovo protein expression, we have examined the expression of a cytokine(hGM-CSF; FIG. 4) and beta-galactosidase (LacZ; FIG. 5) in small-scalebatch culture of W3110 hns205. Expression of both proteins was observedin indole-treated cultures.

It is known that Rcd-induced quiescence can be achieved in an rnc14mutant host as well as in an hns205 mutant (Summers & Rowe, 1997). Wehave shown that 2 mM indole will induce quiescence in an rnc14 mutant ofW3110 (FIG. 6).

We compared the response of cultures W3110 hns205 to Rcd over-expressionor to the addition of indole (2 mM) to the growth medium (both at 42°C.). The two treatments caused the cultures to enter a quiescent statewith similar growth kinetics (FIG. 7).

Materials and Methods, and Further Discussion of Results

For the experimentation for which results are shown in FIG. 1, E. coliBW25113 (Datsenko & Wanner, 2000)was grown overnight at 37° C. inL-broth (Kennedy, 1971). The stationary phase culture was used toinoculate a series of L-broth cultures supplemented with indole (0-6 mM)and their growth was monitored over the next 7 hours.

The addition of 1 mM indole to the culture medium had no effect upongrowth. Concentrations of 2 or 3 mM caused a slowing of the growth ratewhile 4, 5 or 6 mM resulted in almost complete inhibition of growth.

For the experimentation for which results are shown in FIG. 2 a and FIG.2 b, W3110 and W3110hns-205 (Mukherjee et al., 2004) were cultured at37° C. in tryptone water broth (Oxoid) supplemented with 0, 2 or 4 mMindole. W3110 showed a reduced growth rate in response to 2 mM indolealthough the culture eventually reached a density similar to that of theindole-free control. An indole concentration of 4 mM completelyinhibited growth of W3110 (FIG. 2 a).

The response of W3110hns-205 to 2 mM indole was distinct from that ofits wild-type parent. The culture showed a reduction in growth rate overapprox. 6 hours, after which there was very little increase in density.The response of the hns205 mutant to 4 mM indole was similar to that ofthe parent strain and almost complete inhibition of growth was observed(FIG. 2 a). Supplementing the growth medium with 0.5 mM tryptophan madeno difference to the response of the cultures to indole.

The response of W3110 hns205 to indole in L-broth was similar to thatobserved in tryptone water. 2 mM indole caused the culture to enter aquiescent state while 4 mM caused complete growth inhibition (FIG. 2 b).

For the experimentation for which results are shown in FIG. 3 a and FIG.3 b, W3110hns205 was cultured in L-broth at 30° C. (FIG. 3 a) or 42° C.(FIG. 3 b). At t=0 each culture was split into two and indole (finalconcentration 2 mM) was added to one part. Indole-treated culturesentered the quiescent state with similar kinetics at both temperatures.The control cultures (no indole) continued to grow until they reachedstationary phase.

For the experimentation for which results are shown in FIG. 4,W3110hns205 containing plasmid pCMT2bompAhGM-CSF^((see below)) (whichcontains the coding sequence for the cytokine hGM-CSF fused to an ompAleader sequence, transcribed from promoter lambda P_(L) under thecontrol of the temperature sensitive repressor cI857) was grown inL-broth at 30° C. to an OD₆₀₀ of 0.36, at which point the culture wastransferred to 42° C. to induce cytokine expression. Simultaneouslyindole was added to the growth medium at final concentrations of 0, 2 or4 mM (FIG. 4 a).

Cytokine production was quantified by ELISA of total cell lysates andassay data were normalised for culture density (FIG. 4 b). Culturestreated with 4 mM indole (which had stopped growing immediately uponindole addition) produced no hGM-CSF. The addition of 2 mM indoleestablished a quiescent state and in these cultures hGM-CSF increasedrapidly for the first hour although the protein concentration declinedthereafter. In the control culture (no indole), hGM-CSF increased in thefirst hour before declining. The peak concentration was less than halfthat seen in the quiescent culture.

Plasmid pCMT2bompAhGM-CSF was generated as follows: The hGM-CSF gene,with an ompA leader sequence, expressed from the lambdaP_(L) promoter,was excised from plasmid A235 (supplied by Amy Isaacson, R&D Systems) bya SalI and BamHI double digest. The approx. 800 bp product was insertedinto pCMT2b (Mukherjee et al., 2004) also cut with SalI and BamHI.

For the experimentation for which results are shown in FIGS. 5 a and 5b, W3110hns-205 containing pCMT2blacZ (a plasmid which contains thecoding sequence for beta-galactosidase, transcribed from promoter lambdaP_(L) under the control of the temperature sensitive repressor cI857)was grown in L-broth at 30° C. to OD₆₀₀=0.15. The culture was thendivided into two and the temperature was increased to 42° C. to inducelacZ expression. Indole (2 mM) was added immediately to one subcultureto induce a quiescent state, while the other was an indole-free control.Beta-galactosidase activities were measured for 24 hours after thetemperature shift.

The growth of the two cultures at 42° C. is shown in FIG. 5 a. Theindole-treated culture of W3110hns-205 pCMT2blacZ entered a quiescentstate, reaching a final OD₆₀₀ of 0.78, while the control culturecontinued to grow and reached a final OD₆₀₀ of 2.9. For both cultures aninduction of beta-galactosidase expression was monitored at 42° C. (FIG.5 b). For the control culture beta-galactosidase activity increasedrapidly for two hours after transfer to 42° C., reaching just under30,000 MU before decreasing to a level of 12,000 MU after 24 h. In thequiescent culture beta-galactosidase showed a more gradual and sustainedincrease, reaching 27,000 MU after 24 h.

For the experimentation for which results are shown in FIG. 6, a cultureof W3110 rnc-14 was grown in L-broth at 37° C. to a density ofOD₆₀₀=0.15. The culture was divided into two (t=0) and indole (2 mM) wasadded to one of the sub-cultures. Growth of the cultures was measuredover the following 27 hours. While the control culture eventuallyreached stationary phase at OD₆₀₀=2, the indole-treated culture entereda non-growing state after approximately 7 hours and remained at adensity of OD₆₀₀=1 (approx.). Thus an rnc-14 mutant strain entersquiescence in response to indole with kinetics similar to an hns205mutant.

The experiment for which results are shown in FIG. 7 compared the growthkinetics of Rcd-induced and indole-induced quiescent cultures. Culturesof W3110hns-205 pcIts⁸⁵⁷ (Remaut et al., 1983) transformed with eitherpRcd1 (Rcd expression plasmid; Mukherjee et al., 2004)) or pUCdeltalacZ(control plasmid; no Rcd) were grown in L-broth at 30° C. for 90minutes, then 2×5 ml aliquots of each culture were transferred to 50 mlconical flasks, pre-warmed to 42° C. (t=0). The temperature shiftinduces Rcd expression in cultures containing plasmid pRcdl but not incultures containing pUCdeltalacZ. For each pair of cultures, indole(final concentration 2 mM) was added to one of them. IPTG was added toall cultures so that the induction of chromosomal LacZ could bemonitored. Culture density was monitored over the following 24 h.

W3110hns-205 pcIts⁸⁵⁷ pUCdeltalacZ (no indole, no Rcd) grew normally tostationary phase. W3110hns205 pcIts⁸⁵⁷ pRcd1 and W3110hns-205 pcIts⁸⁵⁷plus indole (2 mM) both entered a quiescent state, showing similarkinetics and final cell densities. W3110hns205 pcIts⁸⁵⁷ pRcd1 plusindole (2 mM) also entered a quiescent state but more rapidly andreached a lower final density.

pUCdeltalacZ is pUC18 (Yanisch-Perron et al., 1985) with the lacZfragment and MCS (between co-ordinates 306 and 628) removed by PvuIIdigestion followed by self-ligation.

The Effect of Indole Compounds on the Induction of Quiescence IN E. ColiW3110HNS-205::TN10

As described above, the addition of indole to the culture medium of E.coli W3110hns-205::Tn10 induces quiescence. We examined the effect ofeight indole compounds on growth of E. coli in broth culture and foundthat isoquinoline and 3-β-indoleacrylic acid had very similar effects toindole.

Quinoline, indoline and tryptamine caused some inhibition of growth buttheir effect was less severe than indole. Pyrrole and indole acetic acidwere not observed to have a significant effect on growth.

Materials and Methods

A single E. coli colony (W3110 hns-205::Tn10) was picked from an L-agarplate containing tetracycline (10 μg ml⁻¹) and inoculated into 10 mlL-broth. This was grown overnight in a shaking incubator at 37° C. 200μl of overnight culture was inoculated into 20 ml L-broth in a 50 mlconical flask, and grown at 37° C. in a shaking water bath to an opticaldensity at 600 nm (OD₆₀₀) of between 0.2 and 0.3. Two ml of this culturewas inoculated into 18 ml pre-warmed L-broth containing tetracycline (10μg ml⁻¹) to which had been added an appropriate volume of indole,isoquinoline, indoline, tryptamine, quinoline, or pyrrole stock solution(0.5 M in ethanol), IAA stock solution (0.5 M in water),3-β-indoleacrylic acid stock solution (0.125 M in ethanol) or1-acetylindoline stock solution (0.25 M in ethanol) to give a finalconcentration of 3 mM. (3 mM indole is the minimum concentration whichcauses immediate growth inhibition of E. coli in L-broth containingtetracycline.) Appropriate control experiments were performed to ensurethat the ethanol introduced as the solvent for indole and relatedcompounds (final conc. either 0.6 or 2.4%) was not affecting the growthof the culture.

See Table 1 for compound structures.

Results

Table 2 summarises the effect of indole and related compounds (each at afinal concentration of 3 mM) on the growth of E. coli W3110 hns205 inL-broth containing tetracycline. Detailed growth data are given in FIGS.8-11. Isoquinoline (FIG. 8) and 3-β-indoleacrylic acid (FIG. 11) hadvery similar effects to indole. Quinoline (FIG. 10) and 1-acetylindoline(FIG. 11) showed substantial growth inhibition, but less severe thanthat shown by indole. Indoline (FIG. 8) and tryptamine (FIG. 9)exhibited slight growth inhibition, while pyrrole (FIG. 10) had noobserved inhibitory effect at the concentration employed, and IAA (FIG.9) caused slight stimulation of growth. The latter observation isconsistent with reports in the literature that IAA assists the bacterialstress response (Bianco et al., 2006).

CONCLUSION

The results indicate that indole compounds such as isoquinoline,3-β-indoleacrylic acid, quinoline and 1-acetylindoline show significantinhibitory effects in the growth of E. coli W3110 hns205 and thereforeprovide alternatives to indole for the establishment of the quiescentstate in broth culture. Other alternative compounds may be identifiedsimply by following the testing regime described above.

TABLE 1 Structures of indole-related compounds tested for their effecton the growth of E. coli W3110hns-205::Tn10 isoquinoline

indoline

tryptamine

indole-3-acetic acid (IAA)

quinoline

pyrrole

3-β-indoleacrylic acid

1-acetylindoline

TABLE 2 Summary of the effect of indole and related compounds onW3110hns-205::Tn10. Stock Growth Compound Solution inhibition^(a) indole0.5 M in +++ ethanol isoquinoline 0.5 M in +++ ethanol indoline 0.5 Min + ethanol tryptamine 0.5 M in + ethanol IAA 0.5 M in −^(b) waterquinoline 0.5 M in ++ ethanol pyrrole 0.5 M in − ethanol3-β-indoleacrylic 0.125 M in +++ acid ethanol 1-acetylindoline 0.25 M in++ ethanol ^(a)Semi-quantitative estimate of growth inhibition: +++indicates that inhibition was equivalent to that seen with indole; −indicates that there was no detectable effect on growth. ^(b)Theaddition of IAA resulted in slight growth stimulation.

REFERENCES

-   Baneyx (1999) Current Opinion in Biotechnology 10, 411-421.-   Bianco et al. Arch Microbiol 185, 373-382.-   Datsenko & Wanner (2000) PNAS 97, 6640-6645.-   Flickinger & Rouse (1993) Biotechnology Progress 9, 555-572.-   Hirakawa et al. (2005) Molecular Microbiology 55, 1113-1126.-   Kaprelyants et al. (1993) FEMS Microbiology Reviews 104, 271-286.-   Kennedy (1971) J Bacteriol 108, 10-19.-   Matin (1992) J Appl Bacteriol Symp Suppl 73, 49S-57S.-   Mukherjee et al. (2004) Applied and Environmental Microbiology 70,    3005-3012.-   Patient & Summers (1993) Mol Microbiol 8, 1089-1095.-   Remaut et al. (1983) Gene 22, 103-113.-   Rowe & Summers (1999) Applied and Environmental Microbiology 65,    2710-2715.-   Sharpe et al. (1999) Microbiology 145, 2135-2144.-   Summers & Rowe (1997 WO97/34996-   Tunner et al. (1992) Biotechnology and Bioengineering 40, 271-279.-   Wang et al. (2001) J Bacteriol 183, 4210-4216.-   Yanisch-Perron et al. (1985) Gene 33, 103-119.

1. A method of producing quiescent cells, the method comprising treatingbacterial cells with at least 1 mM indole, wherein the cells are hns⁻bacterial cells.
 2. A method according to claim 1 wherein the cells havea rnc mutation.
 3. A method according to claim 1 wherein the cells areE. coli.
 4. A method according to claim 1 wherein the cells are growingin broth culture prior to treatment with indole.
 5. A method accordingto claim 1 wherein the cells contain an extra-chromosomal heterologousgene prior to treatment with indole, the method comprising causing orallowing expression of the heterologous gene in the quiescent cells. 6.A method according to claim 5 wherein the heterologous gene is undercontrol of an inducible promoter, and expression of the heterologousgene is induced after the cells enter quiescence.
 7. A method accordingto claim 5 wherein the heterologous gene is under control of a promoterthat is up-regulated in response to indole.
 8. A method according toclaim 1 comprising introducing a heterologous gene into the quiescentcells and causing or allowing expression from the heterologous gene inthe quiescent cells.
 9. A method for producing a gene product frombacterial cells containing an extra-chromosomal heterologous geneencoding the gene product, the method comprising: growing in brothculture bacterial cells containing the heterologous gene; treating thecells with at least 1 mM indole to induce quiescence in the cell; andcausing or allowing expression of the heterologous gene in the quiescentcells, wherein the cells are hns⁻ bacterial cells.
 10. A methodaccording to claim 9 comprising, prior to growing the cells in brothculture, a step of introducing the heterologous gene into the bacterialcells.
 11. A method for producing a gene product from bacterial cellscontaining an extra-chromosomal heterologous gene encoding the geneproduct, the method comprising: growing in broth culture bacterial cell;treating the cells with at least 1 mM indole to induce quiescence in thecell; introducing into the quiescent cells the heterologous gene; andcausing or allowing expression of the heterologous gene in the quiescentcells, wherein the cells are hns⁻ bacterial cells.
 12. A methodaccording to claim 5 wherein expression from the heterologous generesults in production of a gene product encoded by the heterologousgene, the method further comprising isolating the gene product from thecells, or cell culture.
 13. A method according to claim 5 wherein theheterologous gene encodes a gene product that is toxic for the cells, oradversely affects viability, cell growth and/or cell division of thecells.
 14. A method according to claim 12 further comprising modifyingthe gene product and/or formulating the gene product into a compositionwhich includes at least one additional component.
 15. A method ofmonitoring expression from an extrachromosomal gene of interest, themethod comprising: introducing the gene into bacterial cells; growingthe cells in broth culture; treating the cells with at least 1 mM indoleto induce quiescence in the cells; causing or allowing expression fromthe gene of interest; and determining expression in the cell, whereinthe introducing of the gene into the cells is before, along with orafter the treating with indole to induce quiescence, and wherein thecells are hns⁻ bacterial cells.
 16. A method according to claim 15wherein mRNA production is determined.
 17. A method according to claim15 wherein polypeptide production is determined.
 18. A method accordingto claim 17 wherein polypeptide production is determined by observingpolypeptide on an electrophoretic gel.
 19. A method for amplifying thecopy number of an extrachromosomal gene of interest in bacterial cells,the method comprising: introducing the gene into bacterial cells;growing the cells in broth culture; and treating the cells with at least1 mM indole to induce quiescence, wherein the introducing of the geneinto the cells is before, along with or after the treating with indoleto induce quiescence, and wherein the cells are hns⁻ bacterial cells.20. A method according to claim 19 further comprising isolating theextra-chromosomal gene from one or more cells taken from the brothculture.
 21. A method of synchronising cell cycles of cells, the methodcomprising: treating bacterial cells in broth culture with at least 1 mMindole; incubating the cells to achieve quiescence; and removing indolefrom the culture to allow resumption of growth of the cells, wherein thecells are hns⁻ bacterial cells.
 22. A method according to claim 21wherein the cells are E. coli.
 23. A method according to claim 1 whereinthe indole is selected from the group consisting of indole,isoquinoline, 3-β-indoleacrylic acid, quinoline, indoline andtryptamine.
 24. A method according to claim 9 wherein the indole isselected from the group consisting of indole, isoquinoline,3-β-indoleacrylic acid, quinoline, indoline and tryptamine.
 25. A methodaccording to claim 11 wherein the indole is selected from the groupconsisting of indole, isoquinoline, 3-β-indoleacrylic acid, quinoline,indoline and tryptamine.
 26. A method according to claim 15 wherein theindole is selected from the group consisting of indole, isoquinoline,3-β-indoleacrylic acid, quinoline, indoline and tryptamine.
 27. A methodaccording to claim 19 wherein the indole is selected from the groupconsisting of indole, isoquinoline, 3-β-indoleacrylic acid, quinoline,indoline and tryptamine.
 28. A method according to claim 21 wherein theindole is selected from the group consisting of indole, isoquinoline,3-β-indoleacrylic acid, quinoline, indoline and tryptamine.